- Email: [email protected]
Increased expression of growth hormone-releasing hormone in fibrinous inflammation of proliferative diabetic retinopathy
Journal Pre-proof Increased expression of growth hormone-releasing hormone in fibrinous inflammation of proliferative diabetic retinopathy Yong Jie Qin, Sun On Chan, Hong Liang Lin, Yu Qiao Zhang, Bei Ting He, Liang Zhang, Hong Hua Yu, Wai Kit Chu, Chi Pui Pang, Hong Yang Zhang PII:
S0002-9394(20)30059-3
DOI:
https://doi.org/10.1016/j.ajo.2020.02.006
Reference:
AJOPHT 11229
To appear in:
American Journal of Ophthalmology
Received Date: 30 September 2019 Revised Date:
1 February 2020
Accepted Date: 5 February 2020
Please cite this article as: Qin YJ, Chan SO, Lin HL, Zhang YQ, He BT, Zhang L, Yu HH, Chu WK, Pang CP, Zhang HY, Increased expression of growth hormone-releasing hormone in fibrinous inflammation of proliferative diabetic retinopathy, American Journal of Ophthalmology (2020), doi: https:// doi.org/10.1016/j.ajo.2020.02.006. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Inc. All rights reserved.
Abstract PURPOSE: To investigate the involvement of growth hormone-releasing hormone (GHRH)-GH signaling in pathogenesis of proliferative diabetic retinopathy (PDR). DESIGN: Experimental laboratory study. METHODS: Vitreous humor, aqueous humor and serum were obtained from 36 eyes of 36 patients with or without type-2 diabetes from 2017-2019. For histological examination, six fibrovascular membranes were excised from eyes with active PDR. Three fibrovascular membranes were excised from non-diabetic patients with proliferative vitreoretinopathy (PVR) as controls. RESULTS: In PDR, the fibrovascular tissues consisted of a mature region containing fibrocytes, and an immature region populated by abundant polymorphonuclear leukocytes in a fibrinogen meshwork. Clusters of leukocytes were found adhering to the vascular walls. In PVR, no fibrinogen and polymorphonuclear leukocyte was observed in the fibrovascular membranes. The levels of GHRH and GH in PDR were significantly increased (P < 0.001), with 1.8-fold and 72.8-fold in vitreous humor, and 2-fold and 4.9-fold in aqueous humor, respectively, when compared with corresponding levels in controls. No significant difference was detected for insulin-like growth factor-1. Immunohistochemistry showed intense expression of GHRH and its receptor GHRH-R in polymorphonuclear leukocytes, vascular endothelial cells and fibrocytes in fibrovascular membranes of PDR. GHRH staining was not detectable in infiltrating cells within the fibrovascular membrane of PVR. CONCLUSIONS: These findings reveal a possible involvement of GHRH/GHRH-R in fibrinous inflammation that might contribute to the formation of fibrovascular membrane in PDR through mediating activities of leukocytes, vascular endothelial cells and fibrocytes. Targeting GHRH/GHRH-R may be considered as a potential therapeutic approach for the treatment of PDR.
Increased expression of growth hormone-releasing hormone in fibrinous inflammation of proliferative diabetic retinopathy Yong Jie Qin1, Sun On Chan2, Hong Liang Lin1,4, Yu Qiao Zhang1,4, Bei Ting He1,5, Liang Zhang1, Hong Hua Yu1, Wai Kit Chu3, Chi Pui Pang3, and Hong Yang Zhang1* 1
Department of Ophthalmology, Guangdong Eye Institute, Guangdong Academy of Medical Sciences, Guangdong Provincial People's Hospital, Guangzhou, China. Guangzhou, 510080, China
2
School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong
3
Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong
4
Shantou University Medical College, Shantou 515041, China.
5
School of Medicine, South China University of Technology, Guangzhou, 510080, China.
Short Title: GHRH expression in PDR
Corresponding Author:
Prof. HY Zhang Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences. 106 Zhongshan Er Road, Guangzhou, 510080, China. Email: [email protected]
Word Count: 3,199 Table Count: 3 Figure Count: 5 Reference Count: 35
1
Introduction Diabetic retinopathy (DR) is the most common microvascular complication of diabetes mellitus. Pathogenesis of DR is complex and multifactorial, but many molecular and physiologic abnormalities in early diabetic retina are also observed in inflammation.1 Targeting inflammation shows multiple benefits in the treatment of type 2 diabetes mellitus (T2DM) and its associated complications.2 Treatments with anti-vascular endothelial growth factor or steroids have been helpful for intervention of DR, but up to 50% of patients may fail to respond.3 Studying the pathogenesis of DR and mechanisms involved in the progression of non-proliferative diabetic retinopathy to proliferative diabetic retinopathy (PDR) is useful for developing early and successful therapeutic strategies.4 The somatotrope axis, growth hormone-releasing hormone (GHRH)-growth hormone (GH)-insulin-like growth factor-1 (IGF-1), plays a mitogenic role in stimulation of cell survival, proliferation and differentiation.5 GHRH, GH and IGF-1 act on the immune system to augment differentiation of granulocytes from progenitor cells in bone marrow into functionally mature immune cells, and to regulate cytokine production and synthesis of GH and IGF-1.6 Ablation of the signaling axis within macrophages dampens the NLRP3 inflammasome-mediated inflammation.7 Treatment with agonists of GHRH enhances immune cell proliferation and function,8 while antagonists of GHRH suppresses the expression of inflammatory factors such as interleukin-1 beta (IL-1β), nuclear factor kappa-B p65 and cyclooxygenase-2 in benign prostatic hyperplasia.9 Moreover, GHRH receptor (GHRH-R)-deficient mice were found to be less susceptible to induction of experimental autoimmune encephalomyelitis (EAE),10 but GH treatment in these knockout mice for 32 days restored the original susceptibility to EAE.11 These data suggest that GHRH-GH-IGF-1 pathway plays a regulatory role in the immune system. We reported earlier that in rats with experimentally induced uveitis, expression of GHRH-R was confined to infiltrating macrophages and leukocytes in the aqueous humor, but not to the immune cells in the stroma of iris and ciliary body, suggesting that GHRH-R may mediate maturation and migration of these cells.12 Leukostasis, typified by immune cells trapping in retinal capillaries, is considered as an early event in DR resulted from an increased vascular permeability.3 The trapped immune cells cause direct damage to vascular endothelial cells and physically occlude the capillaries, leading to retinal hypoxia and neovascularization, and eventually inducing the formation of fibrovascular membranes (FVMs).3, 13 Growth of FVMs at vitreoretinal interface is the pathological hallmark of PDR. In this study, we analyzed fibrovascular tissues from patients with PDR in order to examine whether the immune cells observed at early stage of DR could contribute to the development of FVMs in PDR and whether these immune cells express GHRH and its receptor. 2
Methods Study Population This study was approved by the Human Research Ethics Committee of Guangdong Provincial People's Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China and adhered to the tenets of Declaration of Helsinki. Informed consent was obtained from each patient participated in the study. Samples of vitreous, aqueous humor and serum were obtained from 36 eyes of 36 patients with or without T2DM between July 2017 to August 2019. A post-hoc sample size calculation was presented in Supplementary Table 1. Fluids from patients with non-diabetic macular hole, epiretinal membrane or cataract were used as controls. For histological examination, six FVMs were obtained from patients with active PDR during vitrectomy. Three FVMs were taken from patients with non-diabetic proliferative vitreoretinopathy (PVR) due to retinal detachment to serve as controls. Active PDR was defined by the presence of visible large vessels within the proliferative tissues with fresh preretinal hemorrhage.14 Patients were excluded if they had type 1 diabetes, glycated hemoglobin (HbA1c) higher than 10%, recent retinal photocoagulation (less than 6 months), previous vitrectomy, mass vitreous hemorrhage, ocular trauma, uveitis or known systemic inflammatory or hematologic disease. At the beginning of surgery, samples of undiluted vitreous fluid (0.5 – 1.0 mL) and aqueous humor (0.1 – 0.2 mL) were aspirated under standardized conditions before commencing the intraocular infusion of balanced salt solution. In PDR or PVR patients undergoing pars plana vitrectomy, the FVMs were dissected from the retinal surface with a 25-Gauge vitreous cutter.
Histological examination The FVMs of PDR were separately immersed in 10% (w/v) formalin, embedded in paraffin and sectioned for Hematoxylin and Eosin (H&E) staining and immunohistochemistry as previously described.12 The paraffin sections were heated to induce epitope retrieval using a Biocare Medical tissue processor (Walnut Creek, CA) prior to 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma) staining. After blocking with 0.1% bovine serum at room temperature, the sections were washed with Tris borate saline tween-20 (TBST) and then blocked with 5% bovine serum albumin (BSA) for one hour. After incubation with rabbit polyclonal antibody to Fibrinogen (1:1000 dilution, ab34269, Abcam Inc., Cambridge, MA), GHRH-R (1:200 dilution, ab28692) or GHRH (1:200 dilution, ab187512), the slides were washed for 5 minutes in TBST and incubated for one hour with the Horse Radish Peroxidase (HRP) conjugated anti-rabbit secondary antibody diluted with TBST in a ratio of 1:2000. After washing, slides were incubated with DAB and washed immediately under tap water after color development. Slides were then counter-stained with Hematoxylin. Slides were mounted with dibutyl 3
phthalate xylene (DPX) and then observed under a light microscope (Leica Microsystems, Wetzlar, Germany). Three different FVMs from PVR were treated with the same procedures and served as controls.
Enzyme linked immunosorbent assay (ELISA) Vitreous fluid, aqueous humor, and blood samples were centrifuged at 2500 rpm for 15 minutes at 4ºC. The cell-free supernatants were taken for determination of GHRH (human-ELSIA kit, AVIVA Systems Biology, San Diego, CA), GH (DGH00, humanELSIA kit, R&D systems, Minneapolis, MN), and IGF-1 (DG100, human-ELSIA kit, R&D systems, Minneapolis, MN). A portion of the samples was used for total protein assay (Bio-Rad, Hercules, CA). Plasma fibrinogen concentration in EDTA-anticoagulated blood was determined by the Clauss method (Sysmex CA1500; Sysmex Corporation, Kobe, Japan). Glycated hemoglobin (HbA1c) was measured by high-performance liquid chromatography on the D-10 Hemoglobin Testing System (Bio-Rad Laboratories, CA). All experiments were conducted in duplicates.
Statistical analysis Descriptive statistics were calculated for demographic characteristics of patients with macular hole or epiretinal membrane (Control), type 2 diabetes mellitus (T2DM), and PDR. Student’s t-test was used for comparisons of the means between groups. For comparisons of growth factors in ocular fluid and serum, a multivariate regression analysis should be performed but the small sample sizes avoid the use. These data were analyzed by Student’s t-test. Significant difference was defined as P < 0.05. Statistical analyses were conducted using SPSS statistical software package v. 20.0 (IBM).
Results Clinical characteristics of patients A total of 36 patients were recruited: controls (n=12), T2DM (n=12), and PDR (n=12). The demographic characteristics of these patients were summarized in Table 1. There was no significant difference in age and gender among the three groups. However, patients with T2DM and PDR showed a poorer glycaemic control as their mean HbA1c values were significantly higher than that of controls. Of note, the level of fibrinogen was also significantly elevated in both T2DM and PDR. No significant difference was found between T2DM and PDR, in terms of the levels of HbA1c and fibrinogen. 4
Histological examination of the fibrovascular membranes The cellular organization of the FVMs was examined using H&E staining and immunohistochemistry. In H&E staining, the FVMs excised from patients with active PDR could be divided roughly into two regions (Fig. 1A). The mature region was characterized by the presence of abundant differentiated fibrocytes in the stroma, which was richly invested by blood vessels and hemosiderin-laden macrophages (Fig. 1B). The immature region was characterized by the presence of polymorphonuclear leukocytes and macrophage-like cells (Fig. 1A). Most of these polymorphonuclear cells accumulated around the blood vessels, and some were found within the lumen of the vessels (Fig. 1B and 1C). For comparison purposes, we investigated the structure of FVMs from patients with proliferative vitreoretinopathy (PVR). The FVMs from PVR could also be divided into two regions (Fig. 1D). The first region was occupied largely by differentiated fibrocytes. The immature region was occupied by a large number of mononuclear cells, which were rounded in shape with a large nucleus (Fig. 1D, 1E and 1F). In the samples examined, no polymorphonuclear cell was observed in the FVMs from PVR. Moreover, the FVMs from PVR appeared less vascularized and the vessels were intact without obvious sign of cell infiltration (Fig. 1F), when compared with those from PDR. The segregation of the mature region from the immature region was better defined by immunohistochemistry using antibody against fibrinogen. Immunostaining for fibrinogen was localized strongly in immature regions of FVMs from PDR, which were populated with polymorphonuclear cells (Fig. 2A-D). In mature regions, fibrinogen staining was only obvious on the outer layers but not in the stroma of the membrane. High magnification views of the immature region revealed a strong expression of fibrinogen in the polymorphonuclear cells and the lattice of connective tissues in the stroma of the membrane (Fig. 2C and 2D). On the contrary, immunoreactivity for fibrinogen was not observed in the FVMs from PVR (Fig. 2E and 2F). Only a portion of the mononuclear cells expressed a basal level of fibrinogen (Fig. 2G and 2H), and no fibrinogen-rich lattice was observed in the stroma. These findings suggest that a fibrinous inflammation is involved in the development of FVMs in patients with PDR but not with PVR.
Expression of GHRH-GH-IGF1 axis molecules in diabetic retinopathy The levels of GHRH, GH, and IGF-1 in the vitreous, aqueous humor and serum were measured with ELISA and presented in Table 2. Since the intravitreal proteins are elevated to different degrees in patients with PDR due to disruption of the blood-retinal 5
barrier, we corrected the levels of GHRH, GH, and IGF-1 by the levels of total protein in the vitreous or aqueous humor to avoid this confounding factor. The results showed that both GHRH and GH in vitreous humor of the eyes with PDR were significantly increased when compared with the controls (P < 0.001) (Fig. 3A). In aqueous humor, the levels of GHRH and GH in the PDR group were increased significantly (P < 0.001), whilst only GH (P < 0.001) but not GHRH was elevated in patients with T2DM (Fig. 3B). The levels of IGF-1 showed no significant difference in the vitreous and aqueous humor, both in PDR and T2DM patients.
Expression of fibrinogen and GHRH/GHRH-R in fibrovascular membranes To investigate the expression pattern of GHRH and its receptor GHRH-R the fibrovascular tissues, sections of FVMs collected from patients with PDR were processed for immunohistochemistry using antibodies against GHRH, GHRH-R and fibrinogen. Immunostaining for GHRH was observed on the polymorphonuclear cells and vascular endothelial cells that express fibrinogen (Fig. 4A-F). While the mature fibrocytes were stained with GHRH antibody, these cells did not show obvious fibrinogen signals (Fig. 4B). The GHRH-R was localized also on the polymorphonuclear cells, endothelial cells and fibrocytes, similar to GHRH (Fig. 4G-I). Moreover, GHRH and GHRH-R were detected in the polymorphonuclear cells that appeared to penetrate the blood vessels into the surrounding tissue spaces (Fig. 4F and H). In FVMs from PVR, GHRH-R was localized on the fibrocytes and the infiltrated mononuclear cells (Fig. 5A-C). However, expression of GHRH was detected in fibrocytes but not in the infiltrating immune cells (Fig. 5D-F). This unique expression pattern suggests that the signaling of GHRH through its receptor may be involved in the development of FVMs in PDR. The histological findings in FVMs were summarized in Table 3.
Discussion To elucidate the association of fibrovascular membranes with proliferative diabetes retinopathy, we have investigated the organization of FVMs in patients with PDR. The major findings include: (i) the FVMs from PDR was characterized by the presence of fibrin meshwork infiltrated with polymorphonuclear cells, supporting a fibrinous inflammation in PDR, and such fibrin rich network was not seen in the FVMs from proliferative vitreoretinopathy. (ii) Both GHRH and GH, but not IGF-1, were elevated in the vitreous humor in PDR. iii) GHRH and GHRH-R were expressed abundantly in polymorphonuclear leukocytes, vascular endothelial cells and fibrocytes in FVMs from PDR. These findings suggest that GHRH/GHRH-R signaling may be involved in the fibrinous inflammation that leads to the formation of FVMs in PDR. 6
We have isolated FVMs from patients with active PDR and determined whether sustained inflammation may be associated with the development of the membrane. One important finding is the presence of a large number of infiltrating polymorphonuclear cells within a fibrinogen-rich meshwork in the FVMs. Such cytoarchitecture is not observed in FVMs from PVR, which instead are populated by mononuclear leukocytes in a matrix lacking fibrinogen. Infiltration of inflammatory cells has been shown to precede fibrosis in diabetic nephropathy.15 The presence of lymphocytes has been reported in chronic low-grade inflammation that has been implicated in the pathophysiology of DR.16 The polymorphonuclear leukocytes may play a similar role in the formation of FVM in PDR. Moreover, the presence of rich fibrinogen strongly suggests that fibrinous inflammation occurs in the FVM of PDR. A reported study suggested that the development of FVMs may be initiated by neurite outgrowth from neurons and Müller cells.17 Our findings, however, propose a role of fibrinous inflammation in PDR that has not been described. This form of inflammation is marked by an accumulation of secreted fibrinogen and infiltration of leukocytes, particularly neutrophils. Interaction of fibrinogen–monocyte and neutrophil has been shown to promote inflammation to exacerbate tissue damage, which eventually stimulates the ingrowth of fibroblasts and blood vessels at the perivascular space.18 Therefore, accumulation of fibrinogen and leukocytes in local tissues might be responsible for the progressive growth of FVMs in PDR from immature to mature stages. Even before extravasation into the perivascular space, increased fibrinogen in the blood is considered as a high-risk marker for developing vascular inflammatory diseases such as DR.18 In this study, both circulating and secreted forms of fibrinogen are increased in PDR patients. Notably, clusters of polymorphonuclear cells are anchored to the vascular walls and some infiltrating into the tissues of FVMs. These results strongly support the occurrence of inflammation in the pathogenesis of PDR. However, the mechanism that mediates the leukocyte trafficking remains to be elucidated. The role of GH/IGF1 signaling in DR has long been reported.19 GH has welldescribed diabetogenic actions through mediating glucose metabolism and insulin sensitivity.20 It has been demonstrated that GH stimulates directly the proliferation of human retinal microvascular endothelial cells.21 Transgenic mice expressing a GH antagonist gene are resistant to ischemia-induced retinal neovascularization.22 These findings suggest that GH may play a role in PDR by regulating angiogenic activity that leads to the formation of blood vessels. However, blocking activity of GH-R with antagonist, pegvisomant, does not stimulate regression of PDR.23 Inconclusive findings were also reported in studies of octreotide, a synthetic analogue of somatostatin that blocks GH, which was tested for its effects in treatment of diabetic retinopathy. A small randomized controlled trial involving 23 patients over 15 months reported Octreotide 7
treatment produced a reduction in severity of retinopathy.24 However, no significant benefits were observed after 6 years of treatment with ostreotide in patients with moderately severe or severe non-PDR or low risk PDR in two larger randomized controlled trials conducted by Novartis.25 Furthermore, the precise role of bioactive IGF1 in the development of DR remains unsettled. Although an increase of vitreal IGF-1 has been found in patients with DR,26 mRNA levels of IGF-1 in the retina of diabetic patients are lower than that in non-diabetic controls.27 Thus, the increase in intravitreal IGF-1 is probably due to serum diffusion when the blood-retinal barrier is disrupted in response to chronic hyperglycemia.28 In the current study we found that both vitreal GHRH and GH, but not IGF-1, are significantly increased in patients with PDR when compared with non-diabetic patients. Moreover, both GHRH and GHRH-R are localized on the inflammatory cells, vascular endothelial cells and fibrocytes within the FVMs of PDR. Through binding to GHRH-R, GHRH may stimulate the synthesis and secretion of GH that contributes to the elevated level of vitreal GH. High level of GH has been linked to the development of cancer and the microvascular complications associated with diabetes.20 Moreover, increases of aqueous GHRH and GH are also observed in patients with PDR, but no significant difference is observed between PDR and diabetic patients without retinopathy. The proximity of the vitreous humor to the retina may better reflect the pathophysiological processes occurring in the retinal tissues.29 Therefore, the elevated GHRH in the vitreous humor may be relevant to the development of PDR. GHRH has a wide spectrum of extra-pituitary activities, exemplifying by its ability to modulate cell proliferation and differentiation of various cell types.6 We argue that ocular activity of GHRH/GHRH-R might contribute to the diabetic fibrovascular proliferation that is independent of GH/IGF-1 signaling. Fibrinogen serves as an adhesive substrate for a variety of cells including vascular endothelial cells and leukocytes. Receptors CD11b/CD18 on monocyte have been shown to participate in fibrinogen signaling, resulting in local production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and IL-1β.30 However, fibrinogen signaling may mainly be involved in the initial stage of formation of FVMs as it is predominantly present in the immature region rather than the mature region that associates with fibrosis. Notably, the expression of GHRH and GHRH-R is not only confined to the infiltrating polymorphonuclear cells, but also in the vascular endothelial cells and the fibrocytes. We proposed in our earlier study that GHRH-R signaling may affect maturation and migration of immune cells during acute inflammation in anterior segments of the eye.12 GHRH was found to regulate cytokine release from human peripheral blood mononuclear cells and promote differentiation of granulocytes into functional mature immune cells.31 The activated leukocytes can secrete pro-fibrotic cytokines such as TNF-α, IL-13 and transforming growth factor-beta (TGF-β) to initiate the process of fibrosis and remodel the maturation phase.32 Moreover, GHRH/GHRH-R affects fibroblast proliferation as well as their migration and expression of α-smooth 8
muscle actin (αSMA).33 By elevating the expression of αSMA, GHRH regulates the myoepithelial differentiation of stromal fibroblasts.6 Myofibroblasts expressing αSMA are the key cellular mediator of fibrosis, which contribute to the pathologic fibrosis in PDR.34 Taken together, these findings suggest a signaling role of GHRH/GHRH-R that mediates activities of polymorphonuclear leukocytes, vascular endothelial cells and fibrocytes that lead to the formation of FVMs in PDR. Recently, the GHRH/GHRH-R pathway has been implicated in diabetes and its complications.31 Being a survival mediator, GHRH agonist exerted neuroprotective effects in early experimental DR.35 However, in advanced stages of DR with neovascularization and FVM formation, blockage of GHRH/GHRH-R pathway might be beneficial for slowing or attenuating the progression of PDR. Although analysis of ocular fluid and the FVMs provide new insights into the pathogenesis of PDR, this study design was limited by the small sample sizes because most patients with active PDR were presented with severe vitreous haemorrhage. However, the comparative analyses show statistical significance that supports a sufficient statistical power with the existing sample sizes, particularly with P<0.001. Moreover, the quantitative measurements were consistent with the findings in histological examinations. In conclusion, we report evidence for the first time that GHRH/GHRH-R, independent of GH-IGF-1 signaling, is involved in the fibrinous inflammation through mediating activities of leukocytes, vascular endothelial cells and fibrocytes to generate and remodel the formation of FVMs. Our findings suggest a novel action of GHRH/GHRH-R signaling in PDR, which may serve as a potential therapeutic target for treatment.
9
Acknowledgements This work was supported by National Natural Science Foundation of China (grant number 81600752, YJQ), National Natural Science Foundation of Guangdong Province, China (grant number 2018A030313833, HYZ). Science and Technology Program of Guangzhou, China (grant number 201607010390, HYZ). The authors thank the staff of Department of Ophthalmology, Guangdong Academy of Medical Sciences and Guangdong Provincial People's Hospital, who assisted with data collection. The following authors have no financial disclosures: Yong Jie Qin, Sun On Chan, Hong Liang Lin, Yu Qiao Zhang, Bei Ting He, Liang Zhang, Hong Hua Yu, Wai Kit Chu, Chi Pui Pang, and Hong Yang Zhang. The authors attest that they meet the current ICMJE criteria for authorship.
10
References: 1. Tang J, Kern TS. Inflammation in diabetic retinopathy. Prog Retin Eye Res 2011;30(5):343-358. 2. Donath MY. Targeting inflammation in the treatment of type 2 diabetes: time to start. Nat Rev Drug Discov 2014;13(6):465-476. 3. Stitt AW, Curtis TM, Chen M, et al. The progress in understanding and treatment of diabetic retinopathy. Progress in Retinal & Eye Research 2016;51:156-186. 4. Simo R, Hernandez C. Novel approaches for treating diabetic retinopathy based on recent pathogenic evidence. Prog Retin Eye Res 2015;48:160-180. 5. Bodart G, Goffinet L, Morrhaye G, et al. Somatotrope GHRH/GH/IGF-1 axis at the crossroads
between
immunosenescence
and
frailty.
Ann
Ny
Acad
Sci
2015;1351(1):61-67. 6. Kiaris H, Chatzistamou I, Papavassiliou AG, Schally AV. Growth hormone-releasing hormone: not only a neurohormone. Trends Endocrinol Metab 2011;22(8):311-317. 7. Spadaro O, Goldberg E, Camell C, et al. Growth Hormone Receptor Deficiency Protects against Age-Related NLRP3 Inflammasome Activation and Immune Senescence. Cell Rep 2016;14(7):1571-1580. 8. Khorram O, Yeung M, Vu L, Yen SS. Effects of [norleucine27]growth hormonereleasing hormone (GHRH) (1-29)-NH2 administration on the imm une system of aging men and women. J Clin Endocrinol Metab 1997;82(11):3590-3596. 9. Rick FG, Schally AV, Block NL, et al. Antagonists of growth hormone-releasing hormone (GHRH) reduce prostate size in experimental benign pr ostatic hyperplasia. Proc Natl Acad Sci U S A 2011;108(9):3755-3760. 10.Hideto I, Masaharu K, Shinichi K. Cutting edge: Requirement for growth hormonereleasing
hormone
in
the
development
of
experimental
autoimmune
encephalomyelitis. J Immunol 2003;171(6):2769-2772. 11.Rugia S, Pardo CA, Federica G, Schally AV, Roberto S. GH, but not GHRH, plays a role
in
the
development
of
experimental
autoimmune
encephalomyelitis.
Endocrinology 2011;152(10):3803-3810. 12.Qin YJ, Chan SO, Chong KK, et al. Antagonist of GH-releasing hormone receptors alleviates experimental ocular inflammation. Proc Natl Acad Sci U S A 11
2014;111(51):18303-18308. 13.Karlstetter M, Scholz R, Rutar M, Wong WT, Provis JM, Langmann T. Retinal microglia: Just bystander or target for therapy? Prog Retin Eye Res 2014;45:30-57. 14.Hsu YR, Yang CM, Yeh PT. Clinical and histological features of epiretinal membrane
after
diabetic
vitrectomy. Graefes
Arch Clin Exp Ophthalmol
2014;252(3):401-410. 15.Chung AC, Lan HY. Chemokines in renal injury. J Am Soc Nephrol 2011;22(5):802809. 16.Urbančič M, Štunf Š, MilutinovićŽivin A, Petrovič D, GlobočnikPetrovič M. Epiretinal Membrane Inflammatory Cell Density Might Reflect the Activity of Proliferative Diabetic RetinopathyInflammatory Cells and Diabetic Retinopathy. Invest Ophth Vis Sci 2014;55(12):8576-8582. 17.Klaassen I, W. De Vries E, Vogels I, et al. Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranes. Plos One 2017;12:e187304. 18.Davalos D, Akassoglou K. Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol 2012;34(1):43-62. 19.Powell EDU, Frantz AG, Rabkin MT, Field RA. Growth Hormone in Relation to Diabetic Retinopathy. New Engl J Med 1966;275(17):922-925. 20.Lu M, Flanagan JU, Langley RJ, Hay MP, Perry JK. Targeting growth hormone function: strategies and therapeutic applications. Signal Transduct Target Ther 2019;4:3. 21.Rymaszewski Z, Chomczynski RMCA. Human Growth Hormone Stimulates Proliferation of Human Retinal Microvascular Endothelial Cells in vitro. Proc Natl Acad Sci U S A 1991;88(2):617-621. 22.Smith, E. L. Essential Role of Growth Hormone in Ischemia-Induced Retinal Neovascularization. Science 1997;276(5319):1706-1709. 23.Beck RW, Frank RN, Kraker RT, Ellis KJ. The effect of a growth hormone receptor antagonist
drug
on
proliferative
diabetic
retinopathy.
Ophthalmology
2001;108:2266-2272. 24.Grant MB, Mames RN, Fitzgerald C, et al. The efficacy of octreotide in the therapy 12
of severe nonproliferative and early proliferative diabetic retinopathy: a randomized controlled study. Diabetes Care 2000;23(4):504-509. 25.Mohamed Q, Gillies MC, Wong TY. Management of diabetic retinopathy: a systematic review. JAMA 2007;298(8):902-916. 26.Ruberte J, Ayuso E, Navarro M, et al. Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease. J Clin Invest 2004;113(8):1149-1157. 27.Gerhardinger C, McClure KD, Romeo G, Podestà F, Lorenzi M. IGF-I mRNA and Signaling in the Diabetic Retina. Diabetes 2001;50(1):175. 28.Simo R, Lecube A, Segura RM, Garcia AJ, Hernandez C. Free insulin growth factor-I and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy. Am J Ophthalmol 2002;134(3):376-382. 29.Nawaz IM, Rezzola S, Cancarini A, et al. Human vitreous in proliferative diabetic retinopathy: Characterization and translational implications. Prog Retin Eye Res 2019;72:100756. 30.Davalos D, Akassoglou K. Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol 2012;34(1):43-62. 31.Schally AV, Zhang X, Cai R, Hare JM, Granata R, Bartoli M. Actions and Potential Therapeutic Applications of Growth Hormone–Releasing Hormone Agonists. Endocrinology 2019;160(7):1600-1612. 32.Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest 2007;117(3):524-529. 33.Dioufa N, Schally AV, Chatzistamou I, et al. Acceleration of wound healing by growth hormone-releasing hormone and its agonists. Proceedings of the National Academy of Sciences 2010;107(43):18611. 34.El-Asrar AMA, Hertogh GD, Eynde KVD, et al. Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelialto-mesenchymal transition (EndoMT). Exp Eye Res 2015;132(supplement):179-189. 35.Thounaojam MC, Powell FL, Patel S, et al. Protective effects of agonists of growth hormone-releasing hormone (GHRH) in early experimental diabetic retinopathy. Proc Natl Acad Sci U S A 2017; 114 (50):13248-13253.
13
Figure captions: Figure 1. Hematoxylin-eosin staining of the fibrovascular membranes (FVMs). In the FVMs of proliferative diabetic retinopathy (PDR-FVMs), the mature region contained lots of fibrocytes (black arrowhead, A), rich blood vessels (black arrows, A). While in the immature region, there were numerous polymorphonuclear cells (red and green arrowheads, A). Notably, the representative cells of the mature region included some hemosiderin-laden macrophages (green arrow, B), and the polymorphonuclear cells that accumulated and adhered to the vascular walls readily to infiltration (red arrows, B and C). In the FVMs of proliferative vitreoretinopathy (PVR-FVMs), the fibrocytes (black arrowhead, D) were found in the mature region, and lots of infiltrating cells (red arrowhead, D) in the immature region. The infiltrating cells in the immature areas were likely to be monocyte-like cells, but no polymorphonuclear cell was observed (red arrows, E and F). The mature region and immature region of the entire tissue were indicated with a dotted line. V, blood vessel. Figure 2. Immunohistochemistry of fibrinogen in the fibrovascular membranes (FVMs). Surgically excised FVMs were stained with Hematoxylin and Eosin (H&E) and antifibrinogen. HE stain illustrated the structure of the tissue corresponding to its immunoreactive fibrinogen. In the FVMs of proliferative diabetic retinopathy (PDRFVMs), fibrinogen was extensively expressed in the immature region (B), where there were numerous infiltrating polymorphonuclear cells (black arrowhead in A, magnification with 25 µm). But the fibrinogen was rarely detected in the mature region. In the immature area, the polymorphonuclear cells (red arrows, C) were trapped in the hairlike fibrinogen network (green arrows, C). However, In the FVMs of proliferative vitreoretinopathy (PVR-FVMs), the tissues containing infiltrating cells (black arrowheads in E or red arrow in G) and acellular stromal tissues (green arrow, G), were negatively immunoreactive to fibrinogen (F and H). The mature region and immature region of the entire tissue were indicated with a dotted line. Figure 3. Absolute ratio of GHRH, GH, and IGF-1 in vitreous (A) and aqueous humor (B). Compared with the control eyes, the ratio of GHRH and GH to the total proteins in respective vitreous and aqueous humor were increased in PDR. GH levels were also significantly evaluated in T2DM. But there was no difference in IGF-1 concentration in both vitreous and aqueous humor. Data shown were presented as mean ± SD, Student’s t-test, n = 12 in each group. Patients with non-diabetic macular hole or epiretinal membrane were served as controls. T2DM, type 2 diabetes mellitus; PDR, proliferative diabetic retinopathy of patients with T2DM. Figure 4. Expression of fibrinogen and GHRH/GHRH-R in the fibrovascular membranes of PDR (PDR-FVMs). Fibrinogen was mainly detected in the immature region (green arrow, A). Further magnification showed fibrinogen expression was detected 14
predominately in the acellular stromal tissues (green arrows, B), infiltrated cells (red arrow, B), or inside the blood vessels (V, green arrow in C). Mild fibrinogen expression was detected in the vascular endothelial cells (red arrowheads, C), but not in the fibrocytes (black arrows, insert in B). Both GHRH and its receptor GHRH-R were intensively expressed in the infiltrating polymorphonuclear cells or those inside the blood vessels (green arrows). Notably, GHRH and GHRH-R were also strongly expressed in the vascular endothelial cells (red arrowheads, F and I) and fibrocytes (black arrowheads, inserts in E). Figure 5. Expression of fibrinogen and GHRH/GHRH-R in the fibrovascular membranes of PVR (PVR-FVMs). GHRH expression was only detected in the fibrocytes (black arrows, A and B) but not in the infiltrating cells (green arrows, A and C), while GHRH-R expression was observed in both fibrocytes (black arrows, D and E) and infiltrating cells (green arrow, F).
15
Table 1. Clinical characteristics of those included in this study. Variables Age (years)
Control 56.33 ± 13.91
T2DM 58.67 ± 6.26
PDR 55.17 ± 7.87
P-value P = 0.698 b P = 0.803
Gender (M/F)
5/7
6/6
7/5
Duration of diabetes, years
0
6.08 ± 2.57
12.91 ± 4.50
c
HbA1c (%)
5.90 ± 0.35
7.74 ± 1.80
8.43 ± 2.25
a
Fibrinogen (g/L)
3.37 ± 0.76
4.27 ± 0.80
4.70 ± 1.06
a
a
a
P = 0.601 P = 0.436
b
P < 0.001
P = 0.002 P = 0.001
b
P = 0.010 P = 0.002
b
Data shown were presented as mean ± SD, Student’s t-test, n = 12 in each group. aP: DM group compared to control; bP: PDR group compared to control. cP: PDR group compared to T2DM; Patients with non-diabetic macular hole or epiretinal membrane served as controls. T2DM, type 2 diabetes mellitus; PDR, proliferative diabetic retinopathy of patients with T2DM; HbA1c, glycosylated hemoglobin; M, male; F, female.
Table 2. Concentration of GHRH, GH, IGF-1, and total protein in patients included in this study.
Variables GHRH (ng/mL)
Vitreous Control PDR
Aqueous humor Control T2DM 5.70±1.25
PDR
Control
Serum T2DM
8.67±1.78***
13.98±3.52
14.32±4.55
PDR 14.72±5.24
10.88±1.63 24.20±2.63***
4.58±1.70
GH (pg/mL)
0.51±0.42
37.33±2.63***
1.31±0.79 3.91±1.18*** 6.12±1.48***
IGF-1 (ng/mL)
0.62±0.08
0.84±0.17***
0.14±0.07
0.16±0.14
0.16±0.07
37.01±10.20
54.03±15.86
161.99±39.76***
Total Protein (mg/mL)
2.56±0.94
3.19±1.17
2.18±0.75
2.23±0.88
2.10±0.93
50.57±5.79
51.39±6.14
50.85±7.58
822.91±202.14 804.32±226.84 831.60±257.63
Data shown were presented as mean ± SD, Student’s t-test, n = 12 in each group. ***P indicates significant difference with P value less than 0.001: Patients with macular hole or epiretinal membrane served as controls. T2DM, type 2 diabetes mellitus; PDR, proliferative diabetic retinopathy of patients with T2DM; HAc1b, glycosylated hemoglobin; M, male; F, female.
Table 3. Summary of histological examination on the fibrovascular membranes.
PDR-2
45
M
+++
+++
+++
+
PDR-3
51
F
+++
+++
+++
+
Protein expression in the cells Fibrinogen GHRH Fib Vec Pmc Mcy Fib Vec Pmc Mcy Fib + + + + + + + － ± + + + + + + + － ± + + + + + + + － ±
PDR-4
55
F
+++
+++
+++
+
－
±
+
+
+
+
+
+
PDR-5
57
M
+++
+++
+++
+
－
±
+
+
+
+
+
PDR-6
54
F
+++
+++
+++
+
－
±
+
+
+
+
PVR-1
42
F
+++
+
－
+++
－
±
－
－
+
PVR-2
37
F
+++
+
－
+++
－
±
－
－
+
Patients Age Sex PDR-1 55 M
PVR-3
Typical cells in the membranes Fib Vec Pmc Mcy +++ +++ +++ +
GHRH-R Vec Pmc + +
Mcy +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
－
－
+
+
－
+
+
－
－
+
+
－
+
+++ + + + + － － ± － － － － Hematoxylin-eosin staining of the typical cells in the membrane: +++ indicates common detection, + indicates rare
－
+
44
M
+++
+
detection, and －indicates no detection; Immunohistochemistry study of the protein expression: + indicates intensively immunoreactive to the proteins, ± indicates rarely immunoreactive to the proteins, －indicates no immunoreactive to the proteins. PDR, proliferative diabetic retinopathy of patients with type 2 diabetes; PVR, proliferative vitreoretinopathy without diabetes; Fib, fibrocytes; Vec, vascular endothelial cells; Pmc, polymorphonuclear cells; Mcy, monocyte-like cells; GHRH-R, growth hormone releasing hormone receptor; GHRH, growth hormone releasing hormone. M, male; F, female.
Highlights 1. Preretinal fibrovascular membrane in PDR is driven by fibrinous inflammation. 2. GHRH, independent of GH-IGF-1 signaling, is involved in the pathogenesis of PDR. 3. Action of GHRH on leukocytes, endothelial cells and fibrocytes contributes to PDR.
S0002-9394(20)30059-3
DOI:
https://doi.org/10.1016/j.ajo.2020.02.006
Reference:
AJOPHT 11229
To appear in:
American Journal of Ophthalmology
Received Date: 30 September 2019 Revised Date:
1 February 2020
Accepted Date: 5 February 2020
Please cite this article as: Qin YJ, Chan SO, Lin HL, Zhang YQ, He BT, Zhang L, Yu HH, Chu WK, Pang CP, Zhang HY, Increased expression of growth hormone-releasing hormone in fibrinous inflammation of proliferative diabetic retinopathy, American Journal of Ophthalmology (2020), doi: https:// doi.org/10.1016/j.ajo.2020.02.006. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Inc. All rights reserved.
Abstract PURPOSE: To investigate the involvement of growth hormone-releasing hormone (GHRH)-GH signaling in pathogenesis of proliferative diabetic retinopathy (PDR). DESIGN: Experimental laboratory study. METHODS: Vitreous humor, aqueous humor and serum were obtained from 36 eyes of 36 patients with or without type-2 diabetes from 2017-2019. For histological examination, six fibrovascular membranes were excised from eyes with active PDR. Three fibrovascular membranes were excised from non-diabetic patients with proliferative vitreoretinopathy (PVR) as controls. RESULTS: In PDR, the fibrovascular tissues consisted of a mature region containing fibrocytes, and an immature region populated by abundant polymorphonuclear leukocytes in a fibrinogen meshwork. Clusters of leukocytes were found adhering to the vascular walls. In PVR, no fibrinogen and polymorphonuclear leukocyte was observed in the fibrovascular membranes. The levels of GHRH and GH in PDR were significantly increased (P < 0.001), with 1.8-fold and 72.8-fold in vitreous humor, and 2-fold and 4.9-fold in aqueous humor, respectively, when compared with corresponding levels in controls. No significant difference was detected for insulin-like growth factor-1. Immunohistochemistry showed intense expression of GHRH and its receptor GHRH-R in polymorphonuclear leukocytes, vascular endothelial cells and fibrocytes in fibrovascular membranes of PDR. GHRH staining was not detectable in infiltrating cells within the fibrovascular membrane of PVR. CONCLUSIONS: These findings reveal a possible involvement of GHRH/GHRH-R in fibrinous inflammation that might contribute to the formation of fibrovascular membrane in PDR through mediating activities of leukocytes, vascular endothelial cells and fibrocytes. Targeting GHRH/GHRH-R may be considered as a potential therapeutic approach for the treatment of PDR.
Increased expression of growth hormone-releasing hormone in fibrinous inflammation of proliferative diabetic retinopathy Yong Jie Qin1, Sun On Chan2, Hong Liang Lin1,4, Yu Qiao Zhang1,4, Bei Ting He1,5, Liang Zhang1, Hong Hua Yu1, Wai Kit Chu3, Chi Pui Pang3, and Hong Yang Zhang1* 1
Department of Ophthalmology, Guangdong Eye Institute, Guangdong Academy of Medical Sciences, Guangdong Provincial People's Hospital, Guangzhou, China. Guangzhou, 510080, China
2
School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong
3
Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong
4
Shantou University Medical College, Shantou 515041, China.
5
School of Medicine, South China University of Technology, Guangzhou, 510080, China.
Short Title: GHRH expression in PDR
Corresponding Author:
Prof. HY Zhang Department of Ophthalmology, Guangdong Eye Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences. 106 Zhongshan Er Road, Guangzhou, 510080, China. Email: [email protected]
Word Count: 3,199 Table Count: 3 Figure Count: 5 Reference Count: 35
1
Introduction Diabetic retinopathy (DR) is the most common microvascular complication of diabetes mellitus. Pathogenesis of DR is complex and multifactorial, but many molecular and physiologic abnormalities in early diabetic retina are also observed in inflammation.1 Targeting inflammation shows multiple benefits in the treatment of type 2 diabetes mellitus (T2DM) and its associated complications.2 Treatments with anti-vascular endothelial growth factor or steroids have been helpful for intervention of DR, but up to 50% of patients may fail to respond.3 Studying the pathogenesis of DR and mechanisms involved in the progression of non-proliferative diabetic retinopathy to proliferative diabetic retinopathy (PDR) is useful for developing early and successful therapeutic strategies.4 The somatotrope axis, growth hormone-releasing hormone (GHRH)-growth hormone (GH)-insulin-like growth factor-1 (IGF-1), plays a mitogenic role in stimulation of cell survival, proliferation and differentiation.5 GHRH, GH and IGF-1 act on the immune system to augment differentiation of granulocytes from progenitor cells in bone marrow into functionally mature immune cells, and to regulate cytokine production and synthesis of GH and IGF-1.6 Ablation of the signaling axis within macrophages dampens the NLRP3 inflammasome-mediated inflammation.7 Treatment with agonists of GHRH enhances immune cell proliferation and function,8 while antagonists of GHRH suppresses the expression of inflammatory factors such as interleukin-1 beta (IL-1β), nuclear factor kappa-B p65 and cyclooxygenase-2 in benign prostatic hyperplasia.9 Moreover, GHRH receptor (GHRH-R)-deficient mice were found to be less susceptible to induction of experimental autoimmune encephalomyelitis (EAE),10 but GH treatment in these knockout mice for 32 days restored the original susceptibility to EAE.11 These data suggest that GHRH-GH-IGF-1 pathway plays a regulatory role in the immune system. We reported earlier that in rats with experimentally induced uveitis, expression of GHRH-R was confined to infiltrating macrophages and leukocytes in the aqueous humor, but not to the immune cells in the stroma of iris and ciliary body, suggesting that GHRH-R may mediate maturation and migration of these cells.12 Leukostasis, typified by immune cells trapping in retinal capillaries, is considered as an early event in DR resulted from an increased vascular permeability.3 The trapped immune cells cause direct damage to vascular endothelial cells and physically occlude the capillaries, leading to retinal hypoxia and neovascularization, and eventually inducing the formation of fibrovascular membranes (FVMs).3, 13 Growth of FVMs at vitreoretinal interface is the pathological hallmark of PDR. In this study, we analyzed fibrovascular tissues from patients with PDR in order to examine whether the immune cells observed at early stage of DR could contribute to the development of FVMs in PDR and whether these immune cells express GHRH and its receptor. 2
Methods Study Population This study was approved by the Human Research Ethics Committee of Guangdong Provincial People's Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China and adhered to the tenets of Declaration of Helsinki. Informed consent was obtained from each patient participated in the study. Samples of vitreous, aqueous humor and serum were obtained from 36 eyes of 36 patients with or without T2DM between July 2017 to August 2019. A post-hoc sample size calculation was presented in Supplementary Table 1. Fluids from patients with non-diabetic macular hole, epiretinal membrane or cataract were used as controls. For histological examination, six FVMs were obtained from patients with active PDR during vitrectomy. Three FVMs were taken from patients with non-diabetic proliferative vitreoretinopathy (PVR) due to retinal detachment to serve as controls. Active PDR was defined by the presence of visible large vessels within the proliferative tissues with fresh preretinal hemorrhage.14 Patients were excluded if they had type 1 diabetes, glycated hemoglobin (HbA1c) higher than 10%, recent retinal photocoagulation (less than 6 months), previous vitrectomy, mass vitreous hemorrhage, ocular trauma, uveitis or known systemic inflammatory or hematologic disease. At the beginning of surgery, samples of undiluted vitreous fluid (0.5 – 1.0 mL) and aqueous humor (0.1 – 0.2 mL) were aspirated under standardized conditions before commencing the intraocular infusion of balanced salt solution. In PDR or PVR patients undergoing pars plana vitrectomy, the FVMs were dissected from the retinal surface with a 25-Gauge vitreous cutter.
Histological examination The FVMs of PDR were separately immersed in 10% (w/v) formalin, embedded in paraffin and sectioned for Hematoxylin and Eosin (H&E) staining and immunohistochemistry as previously described.12 The paraffin sections were heated to induce epitope retrieval using a Biocare Medical tissue processor (Walnut Creek, CA) prior to 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma) staining. After blocking with 0.1% bovine serum at room temperature, the sections were washed with Tris borate saline tween-20 (TBST) and then blocked with 5% bovine serum albumin (BSA) for one hour. After incubation with rabbit polyclonal antibody to Fibrinogen (1:1000 dilution, ab34269, Abcam Inc., Cambridge, MA), GHRH-R (1:200 dilution, ab28692) or GHRH (1:200 dilution, ab187512), the slides were washed for 5 minutes in TBST and incubated for one hour with the Horse Radish Peroxidase (HRP) conjugated anti-rabbit secondary antibody diluted with TBST in a ratio of 1:2000. After washing, slides were incubated with DAB and washed immediately under tap water after color development. Slides were then counter-stained with Hematoxylin. Slides were mounted with dibutyl 3
phthalate xylene (DPX) and then observed under a light microscope (Leica Microsystems, Wetzlar, Germany). Three different FVMs from PVR were treated with the same procedures and served as controls.
Enzyme linked immunosorbent assay (ELISA) Vitreous fluid, aqueous humor, and blood samples were centrifuged at 2500 rpm for 15 minutes at 4ºC. The cell-free supernatants were taken for determination of GHRH (human-ELSIA kit, AVIVA Systems Biology, San Diego, CA), GH (DGH00, humanELSIA kit, R&D systems, Minneapolis, MN), and IGF-1 (DG100, human-ELSIA kit, R&D systems, Minneapolis, MN). A portion of the samples was used for total protein assay (Bio-Rad, Hercules, CA). Plasma fibrinogen concentration in EDTA-anticoagulated blood was determined by the Clauss method (Sysmex CA1500; Sysmex Corporation, Kobe, Japan). Glycated hemoglobin (HbA1c) was measured by high-performance liquid chromatography on the D-10 Hemoglobin Testing System (Bio-Rad Laboratories, CA). All experiments were conducted in duplicates.
Statistical analysis Descriptive statistics were calculated for demographic characteristics of patients with macular hole or epiretinal membrane (Control), type 2 diabetes mellitus (T2DM), and PDR. Student’s t-test was used for comparisons of the means between groups. For comparisons of growth factors in ocular fluid and serum, a multivariate regression analysis should be performed but the small sample sizes avoid the use. These data were analyzed by Student’s t-test. Significant difference was defined as P < 0.05. Statistical analyses were conducted using SPSS statistical software package v. 20.0 (IBM).
Results Clinical characteristics of patients A total of 36 patients were recruited: controls (n=12), T2DM (n=12), and PDR (n=12). The demographic characteristics of these patients were summarized in Table 1. There was no significant difference in age and gender among the three groups. However, patients with T2DM and PDR showed a poorer glycaemic control as their mean HbA1c values were significantly higher than that of controls. Of note, the level of fibrinogen was also significantly elevated in both T2DM and PDR. No significant difference was found between T2DM and PDR, in terms of the levels of HbA1c and fibrinogen. 4
Histological examination of the fibrovascular membranes The cellular organization of the FVMs was examined using H&E staining and immunohistochemistry. In H&E staining, the FVMs excised from patients with active PDR could be divided roughly into two regions (Fig. 1A). The mature region was characterized by the presence of abundant differentiated fibrocytes in the stroma, which was richly invested by blood vessels and hemosiderin-laden macrophages (Fig. 1B). The immature region was characterized by the presence of polymorphonuclear leukocytes and macrophage-like cells (Fig. 1A). Most of these polymorphonuclear cells accumulated around the blood vessels, and some were found within the lumen of the vessels (Fig. 1B and 1C). For comparison purposes, we investigated the structure of FVMs from patients with proliferative vitreoretinopathy (PVR). The FVMs from PVR could also be divided into two regions (Fig. 1D). The first region was occupied largely by differentiated fibrocytes. The immature region was occupied by a large number of mononuclear cells, which were rounded in shape with a large nucleus (Fig. 1D, 1E and 1F). In the samples examined, no polymorphonuclear cell was observed in the FVMs from PVR. Moreover, the FVMs from PVR appeared less vascularized and the vessels were intact without obvious sign of cell infiltration (Fig. 1F), when compared with those from PDR. The segregation of the mature region from the immature region was better defined by immunohistochemistry using antibody against fibrinogen. Immunostaining for fibrinogen was localized strongly in immature regions of FVMs from PDR, which were populated with polymorphonuclear cells (Fig. 2A-D). In mature regions, fibrinogen staining was only obvious on the outer layers but not in the stroma of the membrane. High magnification views of the immature region revealed a strong expression of fibrinogen in the polymorphonuclear cells and the lattice of connective tissues in the stroma of the membrane (Fig. 2C and 2D). On the contrary, immunoreactivity for fibrinogen was not observed in the FVMs from PVR (Fig. 2E and 2F). Only a portion of the mononuclear cells expressed a basal level of fibrinogen (Fig. 2G and 2H), and no fibrinogen-rich lattice was observed in the stroma. These findings suggest that a fibrinous inflammation is involved in the development of FVMs in patients with PDR but not with PVR.
Expression of GHRH-GH-IGF1 axis molecules in diabetic retinopathy The levels of GHRH, GH, and IGF-1 in the vitreous, aqueous humor and serum were measured with ELISA and presented in Table 2. Since the intravitreal proteins are elevated to different degrees in patients with PDR due to disruption of the blood-retinal 5
barrier, we corrected the levels of GHRH, GH, and IGF-1 by the levels of total protein in the vitreous or aqueous humor to avoid this confounding factor. The results showed that both GHRH and GH in vitreous humor of the eyes with PDR were significantly increased when compared with the controls (P < 0.001) (Fig. 3A). In aqueous humor, the levels of GHRH and GH in the PDR group were increased significantly (P < 0.001), whilst only GH (P < 0.001) but not GHRH was elevated in patients with T2DM (Fig. 3B). The levels of IGF-1 showed no significant difference in the vitreous and aqueous humor, both in PDR and T2DM patients.
Expression of fibrinogen and GHRH/GHRH-R in fibrovascular membranes To investigate the expression pattern of GHRH and its receptor GHRH-R the fibrovascular tissues, sections of FVMs collected from patients with PDR were processed for immunohistochemistry using antibodies against GHRH, GHRH-R and fibrinogen. Immunostaining for GHRH was observed on the polymorphonuclear cells and vascular endothelial cells that express fibrinogen (Fig. 4A-F). While the mature fibrocytes were stained with GHRH antibody, these cells did not show obvious fibrinogen signals (Fig. 4B). The GHRH-R was localized also on the polymorphonuclear cells, endothelial cells and fibrocytes, similar to GHRH (Fig. 4G-I). Moreover, GHRH and GHRH-R were detected in the polymorphonuclear cells that appeared to penetrate the blood vessels into the surrounding tissue spaces (Fig. 4F and H). In FVMs from PVR, GHRH-R was localized on the fibrocytes and the infiltrated mononuclear cells (Fig. 5A-C). However, expression of GHRH was detected in fibrocytes but not in the infiltrating immune cells (Fig. 5D-F). This unique expression pattern suggests that the signaling of GHRH through its receptor may be involved in the development of FVMs in PDR. The histological findings in FVMs were summarized in Table 3.
Discussion To elucidate the association of fibrovascular membranes with proliferative diabetes retinopathy, we have investigated the organization of FVMs in patients with PDR. The major findings include: (i) the FVMs from PDR was characterized by the presence of fibrin meshwork infiltrated with polymorphonuclear cells, supporting a fibrinous inflammation in PDR, and such fibrin rich network was not seen in the FVMs from proliferative vitreoretinopathy. (ii) Both GHRH and GH, but not IGF-1, were elevated in the vitreous humor in PDR. iii) GHRH and GHRH-R were expressed abundantly in polymorphonuclear leukocytes, vascular endothelial cells and fibrocytes in FVMs from PDR. These findings suggest that GHRH/GHRH-R signaling may be involved in the fibrinous inflammation that leads to the formation of FVMs in PDR. 6
We have isolated FVMs from patients with active PDR and determined whether sustained inflammation may be associated with the development of the membrane. One important finding is the presence of a large number of infiltrating polymorphonuclear cells within a fibrinogen-rich meshwork in the FVMs. Such cytoarchitecture is not observed in FVMs from PVR, which instead are populated by mononuclear leukocytes in a matrix lacking fibrinogen. Infiltration of inflammatory cells has been shown to precede fibrosis in diabetic nephropathy.15 The presence of lymphocytes has been reported in chronic low-grade inflammation that has been implicated in the pathophysiology of DR.16 The polymorphonuclear leukocytes may play a similar role in the formation of FVM in PDR. Moreover, the presence of rich fibrinogen strongly suggests that fibrinous inflammation occurs in the FVM of PDR. A reported study suggested that the development of FVMs may be initiated by neurite outgrowth from neurons and Müller cells.17 Our findings, however, propose a role of fibrinous inflammation in PDR that has not been described. This form of inflammation is marked by an accumulation of secreted fibrinogen and infiltration of leukocytes, particularly neutrophils. Interaction of fibrinogen–monocyte and neutrophil has been shown to promote inflammation to exacerbate tissue damage, which eventually stimulates the ingrowth of fibroblasts and blood vessels at the perivascular space.18 Therefore, accumulation of fibrinogen and leukocytes in local tissues might be responsible for the progressive growth of FVMs in PDR from immature to mature stages. Even before extravasation into the perivascular space, increased fibrinogen in the blood is considered as a high-risk marker for developing vascular inflammatory diseases such as DR.18 In this study, both circulating and secreted forms of fibrinogen are increased in PDR patients. Notably, clusters of polymorphonuclear cells are anchored to the vascular walls and some infiltrating into the tissues of FVMs. These results strongly support the occurrence of inflammation in the pathogenesis of PDR. However, the mechanism that mediates the leukocyte trafficking remains to be elucidated. The role of GH/IGF1 signaling in DR has long been reported.19 GH has welldescribed diabetogenic actions through mediating glucose metabolism and insulin sensitivity.20 It has been demonstrated that GH stimulates directly the proliferation of human retinal microvascular endothelial cells.21 Transgenic mice expressing a GH antagonist gene are resistant to ischemia-induced retinal neovascularization.22 These findings suggest that GH may play a role in PDR by regulating angiogenic activity that leads to the formation of blood vessels. However, blocking activity of GH-R with antagonist, pegvisomant, does not stimulate regression of PDR.23 Inconclusive findings were also reported in studies of octreotide, a synthetic analogue of somatostatin that blocks GH, which was tested for its effects in treatment of diabetic retinopathy. A small randomized controlled trial involving 23 patients over 15 months reported Octreotide 7
treatment produced a reduction in severity of retinopathy.24 However, no significant benefits were observed after 6 years of treatment with ostreotide in patients with moderately severe or severe non-PDR or low risk PDR in two larger randomized controlled trials conducted by Novartis.25 Furthermore, the precise role of bioactive IGF1 in the development of DR remains unsettled. Although an increase of vitreal IGF-1 has been found in patients with DR,26 mRNA levels of IGF-1 in the retina of diabetic patients are lower than that in non-diabetic controls.27 Thus, the increase in intravitreal IGF-1 is probably due to serum diffusion when the blood-retinal barrier is disrupted in response to chronic hyperglycemia.28 In the current study we found that both vitreal GHRH and GH, but not IGF-1, are significantly increased in patients with PDR when compared with non-diabetic patients. Moreover, both GHRH and GHRH-R are localized on the inflammatory cells, vascular endothelial cells and fibrocytes within the FVMs of PDR. Through binding to GHRH-R, GHRH may stimulate the synthesis and secretion of GH that contributes to the elevated level of vitreal GH. High level of GH has been linked to the development of cancer and the microvascular complications associated with diabetes.20 Moreover, increases of aqueous GHRH and GH are also observed in patients with PDR, but no significant difference is observed between PDR and diabetic patients without retinopathy. The proximity of the vitreous humor to the retina may better reflect the pathophysiological processes occurring in the retinal tissues.29 Therefore, the elevated GHRH in the vitreous humor may be relevant to the development of PDR. GHRH has a wide spectrum of extra-pituitary activities, exemplifying by its ability to modulate cell proliferation and differentiation of various cell types.6 We argue that ocular activity of GHRH/GHRH-R might contribute to the diabetic fibrovascular proliferation that is independent of GH/IGF-1 signaling. Fibrinogen serves as an adhesive substrate for a variety of cells including vascular endothelial cells and leukocytes. Receptors CD11b/CD18 on monocyte have been shown to participate in fibrinogen signaling, resulting in local production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and IL-1β.30 However, fibrinogen signaling may mainly be involved in the initial stage of formation of FVMs as it is predominantly present in the immature region rather than the mature region that associates with fibrosis. Notably, the expression of GHRH and GHRH-R is not only confined to the infiltrating polymorphonuclear cells, but also in the vascular endothelial cells and the fibrocytes. We proposed in our earlier study that GHRH-R signaling may affect maturation and migration of immune cells during acute inflammation in anterior segments of the eye.12 GHRH was found to regulate cytokine release from human peripheral blood mononuclear cells and promote differentiation of granulocytes into functional mature immune cells.31 The activated leukocytes can secrete pro-fibrotic cytokines such as TNF-α, IL-13 and transforming growth factor-beta (TGF-β) to initiate the process of fibrosis and remodel the maturation phase.32 Moreover, GHRH/GHRH-R affects fibroblast proliferation as well as their migration and expression of α-smooth 8
muscle actin (αSMA).33 By elevating the expression of αSMA, GHRH regulates the myoepithelial differentiation of stromal fibroblasts.6 Myofibroblasts expressing αSMA are the key cellular mediator of fibrosis, which contribute to the pathologic fibrosis in PDR.34 Taken together, these findings suggest a signaling role of GHRH/GHRH-R that mediates activities of polymorphonuclear leukocytes, vascular endothelial cells and fibrocytes that lead to the formation of FVMs in PDR. Recently, the GHRH/GHRH-R pathway has been implicated in diabetes and its complications.31 Being a survival mediator, GHRH agonist exerted neuroprotective effects in early experimental DR.35 However, in advanced stages of DR with neovascularization and FVM formation, blockage of GHRH/GHRH-R pathway might be beneficial for slowing or attenuating the progression of PDR. Although analysis of ocular fluid and the FVMs provide new insights into the pathogenesis of PDR, this study design was limited by the small sample sizes because most patients with active PDR were presented with severe vitreous haemorrhage. However, the comparative analyses show statistical significance that supports a sufficient statistical power with the existing sample sizes, particularly with P<0.001. Moreover, the quantitative measurements were consistent with the findings in histological examinations. In conclusion, we report evidence for the first time that GHRH/GHRH-R, independent of GH-IGF-1 signaling, is involved in the fibrinous inflammation through mediating activities of leukocytes, vascular endothelial cells and fibrocytes to generate and remodel the formation of FVMs. Our findings suggest a novel action of GHRH/GHRH-R signaling in PDR, which may serve as a potential therapeutic target for treatment.
9
Acknowledgements This work was supported by National Natural Science Foundation of China (grant number 81600752, YJQ), National Natural Science Foundation of Guangdong Province, China (grant number 2018A030313833, HYZ). Science and Technology Program of Guangzhou, China (grant number 201607010390, HYZ). The authors thank the staff of Department of Ophthalmology, Guangdong Academy of Medical Sciences and Guangdong Provincial People's Hospital, who assisted with data collection. The following authors have no financial disclosures: Yong Jie Qin, Sun On Chan, Hong Liang Lin, Yu Qiao Zhang, Bei Ting He, Liang Zhang, Hong Hua Yu, Wai Kit Chu, Chi Pui Pang, and Hong Yang Zhang. The authors attest that they meet the current ICMJE criteria for authorship.
10
References: 1. Tang J, Kern TS. Inflammation in diabetic retinopathy. Prog Retin Eye Res 2011;30(5):343-358. 2. Donath MY. Targeting inflammation in the treatment of type 2 diabetes: time to start. Nat Rev Drug Discov 2014;13(6):465-476. 3. Stitt AW, Curtis TM, Chen M, et al. The progress in understanding and treatment of diabetic retinopathy. Progress in Retinal & Eye Research 2016;51:156-186. 4. Simo R, Hernandez C. Novel approaches for treating diabetic retinopathy based on recent pathogenic evidence. Prog Retin Eye Res 2015;48:160-180. 5. Bodart G, Goffinet L, Morrhaye G, et al. Somatotrope GHRH/GH/IGF-1 axis at the crossroads
between
immunosenescence
and
frailty.
Ann
Ny
Acad
Sci
2015;1351(1):61-67. 6. Kiaris H, Chatzistamou I, Papavassiliou AG, Schally AV. Growth hormone-releasing hormone: not only a neurohormone. Trends Endocrinol Metab 2011;22(8):311-317. 7. Spadaro O, Goldberg E, Camell C, et al. Growth Hormone Receptor Deficiency Protects against Age-Related NLRP3 Inflammasome Activation and Immune Senescence. Cell Rep 2016;14(7):1571-1580. 8. Khorram O, Yeung M, Vu L, Yen SS. Effects of [norleucine27]growth hormonereleasing hormone (GHRH) (1-29)-NH2 administration on the imm une system of aging men and women. J Clin Endocrinol Metab 1997;82(11):3590-3596. 9. Rick FG, Schally AV, Block NL, et al. Antagonists of growth hormone-releasing hormone (GHRH) reduce prostate size in experimental benign pr ostatic hyperplasia. Proc Natl Acad Sci U S A 2011;108(9):3755-3760. 10.Hideto I, Masaharu K, Shinichi K. Cutting edge: Requirement for growth hormonereleasing
hormone
in
the
development
of
experimental
autoimmune
encephalomyelitis. J Immunol 2003;171(6):2769-2772. 11.Rugia S, Pardo CA, Federica G, Schally AV, Roberto S. GH, but not GHRH, plays a role
in
the
development
of
experimental
autoimmune
encephalomyelitis.
Endocrinology 2011;152(10):3803-3810. 12.Qin YJ, Chan SO, Chong KK, et al. Antagonist of GH-releasing hormone receptors alleviates experimental ocular inflammation. Proc Natl Acad Sci U S A 11
2014;111(51):18303-18308. 13.Karlstetter M, Scholz R, Rutar M, Wong WT, Provis JM, Langmann T. Retinal microglia: Just bystander or target for therapy? Prog Retin Eye Res 2014;45:30-57. 14.Hsu YR, Yang CM, Yeh PT. Clinical and histological features of epiretinal membrane
after
diabetic
vitrectomy. Graefes
Arch Clin Exp Ophthalmol
2014;252(3):401-410. 15.Chung AC, Lan HY. Chemokines in renal injury. J Am Soc Nephrol 2011;22(5):802809. 16.Urbančič M, Štunf Š, MilutinovićŽivin A, Petrovič D, GlobočnikPetrovič M. Epiretinal Membrane Inflammatory Cell Density Might Reflect the Activity of Proliferative Diabetic RetinopathyInflammatory Cells and Diabetic Retinopathy. Invest Ophth Vis Sci 2014;55(12):8576-8582. 17.Klaassen I, W. De Vries E, Vogels I, et al. Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranes. Plos One 2017;12:e187304. 18.Davalos D, Akassoglou K. Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol 2012;34(1):43-62. 19.Powell EDU, Frantz AG, Rabkin MT, Field RA. Growth Hormone in Relation to Diabetic Retinopathy. New Engl J Med 1966;275(17):922-925. 20.Lu M, Flanagan JU, Langley RJ, Hay MP, Perry JK. Targeting growth hormone function: strategies and therapeutic applications. Signal Transduct Target Ther 2019;4:3. 21.Rymaszewski Z, Chomczynski RMCA. Human Growth Hormone Stimulates Proliferation of Human Retinal Microvascular Endothelial Cells in vitro. Proc Natl Acad Sci U S A 1991;88(2):617-621. 22.Smith, E. L. Essential Role of Growth Hormone in Ischemia-Induced Retinal Neovascularization. Science 1997;276(5319):1706-1709. 23.Beck RW, Frank RN, Kraker RT, Ellis KJ. The effect of a growth hormone receptor antagonist
drug
on
proliferative
diabetic
retinopathy.
Ophthalmology
2001;108:2266-2272. 24.Grant MB, Mames RN, Fitzgerald C, et al. The efficacy of octreotide in the therapy 12
of severe nonproliferative and early proliferative diabetic retinopathy: a randomized controlled study. Diabetes Care 2000;23(4):504-509. 25.Mohamed Q, Gillies MC, Wong TY. Management of diabetic retinopathy: a systematic review. JAMA 2007;298(8):902-916. 26.Ruberte J, Ayuso E, Navarro M, et al. Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease. J Clin Invest 2004;113(8):1149-1157. 27.Gerhardinger C, McClure KD, Romeo G, Podestà F, Lorenzi M. IGF-I mRNA and Signaling in the Diabetic Retina. Diabetes 2001;50(1):175. 28.Simo R, Lecube A, Segura RM, Garcia AJ, Hernandez C. Free insulin growth factor-I and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy. Am J Ophthalmol 2002;134(3):376-382. 29.Nawaz IM, Rezzola S, Cancarini A, et al. Human vitreous in proliferative diabetic retinopathy: Characterization and translational implications. Prog Retin Eye Res 2019;72:100756. 30.Davalos D, Akassoglou K. Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol 2012;34(1):43-62. 31.Schally AV, Zhang X, Cai R, Hare JM, Granata R, Bartoli M. Actions and Potential Therapeutic Applications of Growth Hormone–Releasing Hormone Agonists. Endocrinology 2019;160(7):1600-1612. 32.Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest 2007;117(3):524-529. 33.Dioufa N, Schally AV, Chatzistamou I, et al. Acceleration of wound healing by growth hormone-releasing hormone and its agonists. Proceedings of the National Academy of Sciences 2010;107(43):18611. 34.El-Asrar AMA, Hertogh GD, Eynde KVD, et al. Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelialto-mesenchymal transition (EndoMT). Exp Eye Res 2015;132(supplement):179-189. 35.Thounaojam MC, Powell FL, Patel S, et al. Protective effects of agonists of growth hormone-releasing hormone (GHRH) in early experimental diabetic retinopathy. Proc Natl Acad Sci U S A 2017; 114 (50):13248-13253.
13
Figure captions: Figure 1. Hematoxylin-eosin staining of the fibrovascular membranes (FVMs). In the FVMs of proliferative diabetic retinopathy (PDR-FVMs), the mature region contained lots of fibrocytes (black arrowhead, A), rich blood vessels (black arrows, A). While in the immature region, there were numerous polymorphonuclear cells (red and green arrowheads, A). Notably, the representative cells of the mature region included some hemosiderin-laden macrophages (green arrow, B), and the polymorphonuclear cells that accumulated and adhered to the vascular walls readily to infiltration (red arrows, B and C). In the FVMs of proliferative vitreoretinopathy (PVR-FVMs), the fibrocytes (black arrowhead, D) were found in the mature region, and lots of infiltrating cells (red arrowhead, D) in the immature region. The infiltrating cells in the immature areas were likely to be monocyte-like cells, but no polymorphonuclear cell was observed (red arrows, E and F). The mature region and immature region of the entire tissue were indicated with a dotted line. V, blood vessel. Figure 2. Immunohistochemistry of fibrinogen in the fibrovascular membranes (FVMs). Surgically excised FVMs were stained with Hematoxylin and Eosin (H&E) and antifibrinogen. HE stain illustrated the structure of the tissue corresponding to its immunoreactive fibrinogen. In the FVMs of proliferative diabetic retinopathy (PDRFVMs), fibrinogen was extensively expressed in the immature region (B), where there were numerous infiltrating polymorphonuclear cells (black arrowhead in A, magnification with 25 µm). But the fibrinogen was rarely detected in the mature region. In the immature area, the polymorphonuclear cells (red arrows, C) were trapped in the hairlike fibrinogen network (green arrows, C). However, In the FVMs of proliferative vitreoretinopathy (PVR-FVMs), the tissues containing infiltrating cells (black arrowheads in E or red arrow in G) and acellular stromal tissues (green arrow, G), were negatively immunoreactive to fibrinogen (F and H). The mature region and immature region of the entire tissue were indicated with a dotted line. Figure 3. Absolute ratio of GHRH, GH, and IGF-1 in vitreous (A) and aqueous humor (B). Compared with the control eyes, the ratio of GHRH and GH to the total proteins in respective vitreous and aqueous humor were increased in PDR. GH levels were also significantly evaluated in T2DM. But there was no difference in IGF-1 concentration in both vitreous and aqueous humor. Data shown were presented as mean ± SD, Student’s t-test, n = 12 in each group. Patients with non-diabetic macular hole or epiretinal membrane were served as controls. T2DM, type 2 diabetes mellitus; PDR, proliferative diabetic retinopathy of patients with T2DM. Figure 4. Expression of fibrinogen and GHRH/GHRH-R in the fibrovascular membranes of PDR (PDR-FVMs). Fibrinogen was mainly detected in the immature region (green arrow, A). Further magnification showed fibrinogen expression was detected 14
predominately in the acellular stromal tissues (green arrows, B), infiltrated cells (red arrow, B), or inside the blood vessels (V, green arrow in C). Mild fibrinogen expression was detected in the vascular endothelial cells (red arrowheads, C), but not in the fibrocytes (black arrows, insert in B). Both GHRH and its receptor GHRH-R were intensively expressed in the infiltrating polymorphonuclear cells or those inside the blood vessels (green arrows). Notably, GHRH and GHRH-R were also strongly expressed in the vascular endothelial cells (red arrowheads, F and I) and fibrocytes (black arrowheads, inserts in E). Figure 5. Expression of fibrinogen and GHRH/GHRH-R in the fibrovascular membranes of PVR (PVR-FVMs). GHRH expression was only detected in the fibrocytes (black arrows, A and B) but not in the infiltrating cells (green arrows, A and C), while GHRH-R expression was observed in both fibrocytes (black arrows, D and E) and infiltrating cells (green arrow, F).
15
Table 1. Clinical characteristics of those included in this study. Variables Age (years)
Control 56.33 ± 13.91
T2DM 58.67 ± 6.26
PDR 55.17 ± 7.87
P-value P = 0.698 b P = 0.803
Gender (M/F)
5/7
6/6
7/5
Duration of diabetes, years
0
6.08 ± 2.57
12.91 ± 4.50
c
HbA1c (%)
5.90 ± 0.35
7.74 ± 1.80
8.43 ± 2.25
a
Fibrinogen (g/L)
3.37 ± 0.76
4.27 ± 0.80
4.70 ± 1.06
a
a
a
P = 0.601 P = 0.436
b
P < 0.001
P = 0.002 P = 0.001
b
P = 0.010 P = 0.002
b
Data shown were presented as mean ± SD, Student’s t-test, n = 12 in each group. aP: DM group compared to control; bP: PDR group compared to control. cP: PDR group compared to T2DM; Patients with non-diabetic macular hole or epiretinal membrane served as controls. T2DM, type 2 diabetes mellitus; PDR, proliferative diabetic retinopathy of patients with T2DM; HbA1c, glycosylated hemoglobin; M, male; F, female.
Table 2. Concentration of GHRH, GH, IGF-1, and total protein in patients included in this study.
Variables GHRH (ng/mL)
Vitreous Control PDR
Aqueous humor Control T2DM 5.70±1.25
PDR
Control
Serum T2DM
8.67±1.78***
13.98±3.52
14.32±4.55
PDR 14.72±5.24
10.88±1.63 24.20±2.63***
4.58±1.70
GH (pg/mL)
0.51±0.42
37.33±2.63***
1.31±0.79 3.91±1.18*** 6.12±1.48***
IGF-1 (ng/mL)
0.62±0.08
0.84±0.17***
0.14±0.07
0.16±0.14
0.16±0.07
37.01±10.20
54.03±15.86
161.99±39.76***
Total Protein (mg/mL)
2.56±0.94
3.19±1.17
2.18±0.75
2.23±0.88
2.10±0.93
50.57±5.79
51.39±6.14
50.85±7.58
822.91±202.14 804.32±226.84 831.60±257.63
Data shown were presented as mean ± SD, Student’s t-test, n = 12 in each group. ***P indicates significant difference with P value less than 0.001: Patients with macular hole or epiretinal membrane served as controls. T2DM, type 2 diabetes mellitus; PDR, proliferative diabetic retinopathy of patients with T2DM; HAc1b, glycosylated hemoglobin; M, male; F, female.
Table 3. Summary of histological examination on the fibrovascular membranes.
PDR-2
45
M
+++
+++
+++
+
PDR-3
51
F
+++
+++
+++
+
Protein expression in the cells Fibrinogen GHRH Fib Vec Pmc Mcy Fib Vec Pmc Mcy Fib + + + + + + + － ± + + + + + + + － ± + + + + + + + － ±
PDR-4
55
F
+++
+++
+++
+
－
±
+
+
+
+
+
+
PDR-5
57
M
+++
+++
+++
+
－
±
+
+
+
+
+
PDR-6
54
F
+++
+++
+++
+
－
±
+
+
+
+
PVR-1
42
F
+++
+
－
+++
－
±
－
－
+
PVR-2
37
F
+++
+
－
+++
－
±
－
－
+
Patients Age Sex PDR-1 55 M
PVR-3
Typical cells in the membranes Fib Vec Pmc Mcy +++ +++ +++ +
GHRH-R Vec Pmc + +
Mcy +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
－
－
+
+
－
+
+
－
－
+
+
－
+
+++ + + + + － － ± － － － － Hematoxylin-eosin staining of the typical cells in the membrane: +++ indicates common detection, + indicates rare
－
+
44
M
+++
+
detection, and －indicates no detection; Immunohistochemistry study of the protein expression: + indicates intensively immunoreactive to the proteins, ± indicates rarely immunoreactive to the proteins, －indicates no immunoreactive to the proteins. PDR, proliferative diabetic retinopathy of patients with type 2 diabetes; PVR, proliferative vitreoretinopathy without diabetes; Fib, fibrocytes; Vec, vascular endothelial cells; Pmc, polymorphonuclear cells; Mcy, monocyte-like cells; GHRH-R, growth hormone releasing hormone receptor; GHRH, growth hormone releasing hormone. M, male; F, female.
Highlights 1. Preretinal fibrovascular membrane in PDR is driven by fibrinous inflammation. 2. GHRH, independent of GH-IGF-1 signaling, is involved in the pathogenesis of PDR. 3. Action of GHRH on leukocytes, endothelial cells and fibrocytes contributes to PDR.