The effects of VEGF on survival of a random flap in the rat: examination of various routes of administration

Br#ish Journal of Plastie Surgery (2000), 53, 234-239 9 2000 The British Association of Plastic Surgeons DOI: 10.1054/bjps.1999.3315

BRITISH

JOURNAL

PLASTIC

SURGERY

The effects of VEGF on survival of a random flap in the rat: examination of various routes of administration Z. Kryger, E Zhang*, T. Dogan]-, C. Chengt, W. C. Lineaweaver* and H. J. Buncket

Division of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford, California; *Division of Plastic Surgery, University of Mississippi Medical Center, Jackson, Mississipp# and 7"Mierosurgical Replantation Transplantation Department, California Pacific Medical Center, Davies Campus', San Fransisco, California, USA SUMMARY. The purpose of the present study was to determine the effects of vascular endothelial growth factor (VEGF) on survival of a full thickness random pattern, McFarlane musculocutaneous flap in the rat. In addition, this study examined a number of different methods of VEGF delivery in an attempt to determine the most effective route of administration. A 2 x 8 cm full thickness dorsal flap with the pedicle remaining attached at the anterior end was elevated in 72 male Sprague-Dawley rats. The rats were randomised into six groups and immediately received the following treatment: Group I (n = 12): systemic VEGF injection into the femoral vein (50 Bxg/ml); Group II (n = 10): multiple systemic VEGF injections at 0, 24 and 48 h post flap elevation (50 ~tg/ml);Group III (n = 12): subdermal VEGF injection into the flap (1 ~tg/ml); Group IV (n = 12): subfascial VEGF injections into the recipient bed (1 ~tg/ml); Group V (n = 10): topical VEGF onto the recipient bed (1 ~tg/ml); Group VI (n = 16): control group with no treatment. Following 5 days recovery, the area of flap survival was measured. Mean flap survival ranged from 91% in Group II to 78% in Group V, and was significantly greater in all experimental groups (P < 0.001 for Groups I-IV and P < 0.05 for Group V) as compared to the control group (mean survival of 66%). The only significant difference between the experimental groups was between the mean survival in Group II and Group V (P < 0.05). Histological analysis demonstrated a qualitatively greater amount of granulation tissue and neovascularisation in the experimental groups. These results support the notion that VEGF rescues tissue at risk of hypoxic damage by inducing angiogenesis, and the use of growth factors such as VEGF holds promise as a method of increasing skin viability. 9 2000 The British Association of Plastic Surgeons

Keywords: vascular endothelial growth factor, random pattern flap survival, route of administration, experimental rat model.

Despite a greater understanding of the mechanisms underlying flap necrosis and the advances in surgical technique, skin flap necrosis remains a problem in the field of plastic and reconstructive surgery. The causes of necrosis can usually be traced to alterations in the haemodynamics of the flap: insufficient arterial flow, inadequate venous drainage, or a combination of the two. In an attempt to increase skin flap viability, the effects of a number of growth factors that induce neovascularisation have been examined. Several of these factors, most notably transforming-, fibroblast- and endothelial cell-growth factor have demonstrated a marked ability to improve skin flap survival. 14 These factors have already shown considerable promise in the fields of vascular and cardiovascular medicine for treating prolonged and secondary ischaemia. 5-7 Vascular endothelial growth factor (VEGF) is the most potent of these angiogenic agents. Its receptors are found solely on endothelial cells,8,9 and it is expressed under conditions of hypoxia1~ and following endothelial damage. 13 VEGF has been shown to increase both blood flow and skin flap survival in a random extension of axial pattern skin flaps in the rat, ~4however, very little data are available to support

this finding and to address other questions regarding the effects of VEGF. The purpose of the present study was to determine the effects of VEGF administration on survival of a full thickness random pattern skin flap using a caudally based, dorsal flap in the rat, first described by McFarlane and colleagues. 15 In addition, this study examined a number of different methods of VEGF delivery in an attempt to determine the most effective route of administration.

Materials and methods Seventy-two male Sprague-Dawley rats weighing between 400 and 450 g and cared for under the National Research Council's guidelines for the care and use of laboratory animals were used in this study. The rats were anaesthetised using pentobarbital (NembutalTM) administered by intraperitoneal injection (50 mg/kg). Following induction of general anaesthesia, the dorsal regions were shaved, the animals were placed in a prone position and a rectangle measuring 2 x 8 cm was drawn onto the backs of the rats. 234

Effects of VEGF on random flap survival The 2 x 8 cm full thickness flap, consisting o f skin and the underlying cutaneous maximus muscle and fascial layers, was elevated using blunt dissection. The flaps were elevated with the pedicle remaining attached at the anterior end. Following flap elevation, the rats were randomised into six groups and given the treatment protocol described below. We used the 165 amino acid isoform of recombinant human V E G F (Genentech Inc., S. San Fransisco, CA, USA) suspended in phosphate-buffered saline. All injections were performed using a 30 gauge needle (MPL Technologies Inc., Franklin Park, IL, USA).

Group I: systemic VEGF-single dose (n = 12)

235 above. They were placed in a prone position, photographed and the area of flap survival was calculated by tracing the surviving region onto paper and calculating its area. Viable skin was determined grossly in a blinded fashion, based on appearance, colour and texture. The mean area of flap survival was calculated for each group, and these means were compared to mean survival in the control group. All statistical analysis was performed using a t-test, with a significance level set at P < 0.05. In addition, mean area of survival was compared between the various experimental groups. After calculating the extent of flap survival, the rats were then sacrificed by overdose of pentobarbital and four flaps fi'om each group were removed. These flaps were placed in 10% formalin for 1 week, and then sectioned both longitudinally and transversely to the long axis of the flap. A qualitative analysis of the extent of angiogenesis in the flaps was performed (x 100 magnification) following a standard haematoxylin and eosin stain.

Systemic V E G F was administered by injection into the femoral vein. The rats were placed into a supine position and an oblique groin incision was made on the right side of each animal. The right femoral vein was exposed from its bed between the inguinal ligament and epigastric vein. 1 ml of V E G F (50 ~tg/ml) was injected into the femoral vein over a period of 20 s, after which the incision was closed using 410 nylon sutures.

Results

Group II: systemic VEGF-multiple doses (n = 10)

Gross observations

V E G F was administered in an identical manner to that described above. A second and third administration (same volume and concentration) were given following 24 and 48 h. The third V E G F bolus was injected into the left femoral vein in order to minimise the trauma to which the femoral veins were subjected.

At day 5 postoperatively, the flaps had not shrunk in area, and the regions of survival and necrosis were clearly demarcated in every flap. The surviving skin appeared pink-white, tender, normal in its texture and it bled when cut with a scalpel. In contrast, the necrotic skin was black, rigid and did not bleed when cut. Figure 1 shows examples of two representative flaps, one from an experimental animal and the other from a control, illustrating the differences between the necrotic and viable regions.

Group HL" subdermal VEGF (n = 12) Here 1 ml of V E G F (1 ~tg/ml) was injected into the flap, by administering 0.1 ml of V E G F at 10 equally distributed locations on the thick fascial layer comprising the undersurface of the flap.

Group IV: subfascial VEGF (n = 12) Here 1.5 ml of V E G F (1 gg/ml) was injected into the fascial layer of the recipient bed to which the flap was returned, by administering 0.15 ml of V E G F at 10 equally distributed locations.

Group V." topical VEGF (n = 10) Here 1.5 ml of V E G F (1 ~g/ml) was applied topically onto the fascial layer of the recipient bed to which the flap was returned.

Group VI: control (n = 16) This group received no treatment after elevation of the flap. Following administration of V E G F the flaps were returned to their recipient beds and carefully sutured into place using 4/0 nylon sutures. The rats were returned to individual cages for a period of 5 days, after which they were re-anaesthetised as described

Flap survival The results of the mean area of flap survival are summarised in Table 1, and a comparison of the mean percentage of the surviving area of the six groups is shown in Figure 2. The control group demonstrated a mean viable area of 66%, which is consistent with the results of others. ~6The mean viable percentage for the experimental groups treated with V E G F ranged from 78 to 91%. The surviving area was significantly greater in every group as compared to controls, irrespective of the method of V E G F delivery (P < 0.001 for Groups I-IV; P < 0.05 for Group V), with the most significant results occurring in the group that received multiple intravenous V E G F injections over a period of 72 h. Topical delivery of V E G F onto the recipient bed proved to be the least effective method of delivery, although survival was significantly greater in this group than in controls as indicated above. The only significant difference when comparing the various methods of V E G F delivery was found when Group II, receiving multiple systemic V E G F injections, was compared to Group V, that received V E G F topically (P < 0.05).

236

British Journal of Plastic Surgery

13

A

Figure l - - P h o t o g r a p h s of two representative flaps to illustrate regions of survival and necrosis from the experimental group treated with VEGF by systemic injection (A) and the control group (B). The area of survival in these two rats was 86% and 66%, respectively, which is equal to the mean percent survival of all methods of V E G F delivery and the control group.

Table 1 Mean area and percent survival of flaps from rats treated with VEGF and control rats receiving no treatment (results from postoperative day 5) Group

Mean area of survival in cm2 ( total flap area = 16 cm 2)

Mean survival (%)

14.14 • 1.57

88

14.55 • 1.42

91

14.07 • 1.70

88

13.73 • 1.51

86

12.50 • 2.62

78

10.54 -* 1.17

66

Systemic VEGF-single dose (n = 12) Systemic VEGF-multiple doses (n = 10) Subdermal VEGF (n = 12) Subfascial VEGF (n = 12) Topical V E G F (n = 10) Control (no treatment) ( n = 16) Note: values following '• indicate standard deviations.

100

90 80 70 60 ~Q" 50 40' 30 20 10 0"

p < .001

p < .001

p < .001

]

p < .001

/

systemle- 1 dose

systernir 3 dos~

subdermal

subfasr

topleal

eontrol

Figure 2 - - A comparison of the values for mean percent flap survival between the various methods o f VEGF delivery and the control group. A description of each route of administration is provided in the text.

Although the number of rats in each experimental group was not equal, no animals died during the study, and none of the data were excluded from any of the experimental groups. This was a cooperative study between several investigators, accounting for the unequal number o f rats in each group. Nevertheless, this did not influence the statistical methodology of the study. The control group was made larger than the various experimental groups in order to increase the power of the statistical analysis.

Histologieal findings Representative sections of four flaps from each group were examined. These sections were taken from both necrotic and surviving regions. Necrotic sections were similar in all groups, including controls, and revealed

Effects of VEGF on random flap survival

237

Figure 3--Histological sections of the surviving regions of flaps taken from random flaps treated with VEGF injected systemically. Notice the extensive granulation tissue deep to the cutaneous maximus muscle layer (ml) in the VEGF flap. Scale bar = 100 microns (H & E stain).

evidence of acute inflammation with infiltration of monocytes and neutrophils. Myonecrosis was evident in about 90% of the muscle layer in these sections. These results are consistent with full-thickness ischaemic necrosis. Sections taken from surviving regions of flaps from VEGF-treated animals revealed a consistent pattern of exaggerated amounts of granulation tissue and neovascularisation (Fig. 3). This was most apparent deep to the muscle layer, with evidence of granulation tissue invasion into this layer. The sprouting of new vessels appeared to be primarily of capillary origin. Myonecrosis was evident but limited in its extent. Just superficial to the muscle layer there was a qualitatively greater density of capillaries, arterioles and venuoles than in similar sections taken from control rats. These comparisons are illustrated in Figure 4. In addition, inflammatory infiltrate in the experimentally treated flaps was minimal, and the dermal and epidermal layers were healthy and similar in appearance to normal tissue. Sections taken from viable regions of flaps from the control group failed to show the extent of granulation tissue and angiogenesis apparent in the VEGF-treated rats. The muscular layer seemed to be normal with limited evidence of necrosis and inflammation. As indicated above, there appeared to be fewer vessels lying just superficial to the muscle. The dermal and epidermal layers were healthy and similar in appearance to normal tissue.

Discussion The purpose of the present study was to examine the effects of V E G F on survival of a random pattern flap in the rat, and to determine the most effective method of delivering V E G E The results of this study demonstrated that V E G F significantly improves the area of flap survival. Our findings are consistent with the mechanism by which V E G F is believed to work.

Figure 4 - Histological sections of tile surviving regions of ['laps taken from a rat treated with VEGF delivered by multiple systemic injections (A) and from a control rat (B). Note the difference between the extent of vascularisation superficial to the cutaneous maximus muscle layer. Superficial is towards the top of the figure. Scale bar = 25 microns (H & E stain).

V E G F is an endogenous stimulator of both angiogenesis, ~7,~s a process which is believed to be essential for neovascularisation to occur, and increased vascular permeability, 19,2~hence its original name of vascular permeability factor. It is expressed in developing blood vessels 2~ and its receptors are found exclusively on endothelial cells. 8,9 When tissue is subjected to hypoxia or endothelial damage, expression of the V E G F protein is up-regulated, m-13For instance, microvessels that infiltrate an infarcted region express extremely high levels of m R N A for two high-affinity V E G F receptors, termed flk-1 and flt-1. Viable myocytes adjacent to the region of infarction express significantly elevated levels of V E G E 22 A number of in vivo and in vitro studies in several models of chronic ischaemia have confirmed that V E G F treatment results in neovascularisation, increased blood flow and pressure, improved muscle function and measurable improvements in tissue viability.2S 27 By augmenting angiogenesis beyond the normal level that occurs following skin flap elevation, V E G F administration is able to increase blood flow to the regions of the flap at risk for ischaemic damage. The result of this is a substantial increase in the viability of the flap.

238 Several methods of VEGF delivery were examined in the present study in order to determine which methods effectively increased skin flap viability. Previously, the effects of VEGF on surgical flap survival have only been examined using a single bolus of VEGF delivered directly to the pedicle of the flap. This study demonstrated that VEGF is efficacious when administered in several different manners. The most effective method of delivery in this study was to administer VEGF systemically, in multiple high concentration boluses. Considering the relatively short half-life of VEGF in vivo, of the order of 5-10 min, it is logical to assume that maintaining adequate levels of VEGF at a high concentration should be effective. Nevertheless, the only statistically significant difference between the various routes of administration was between topical and multiple systemic delivery, a finding that appears puzzling due to the rapid metabolic turnover of this factor. The fact that hypoxia induces a four-fold increase in the half-life of VEGF may help to explain the ability of a single administration of VEGF to have such marked effects on flap viability. 28 Furthermore, it has been shown that even brief exposure to VEGF results in evidence of the initiation of angiogenic process within only 10 min. 29 Our finding that VEGF was effective when injected into the recipient bed was not surprising considering the fact that the recipient tissue is known to contribute to the revascularisation of the flap. 3~ The present results, demonstrating that VEGF is effective when delivered by a number of different routes, are consistent with previous studies. We have shown that the most effective method of delivery is by multiple systemic injections which ensure elevated levels of VEGF over a prolonged period. Others have shown that VEGF administered systemically in a single bolus resulted in a significant increase in capillary density, 31 and that controlled delivery of VEGF from 28-day osmotic pumps significantly enhanced angiogenesis and vascular perfusion. 32 However, the importance of determining the side-effects of systemic use of such a powerful factor should not be overlooked in future studies. A number of other methods of VEGF delivery not examined in the present experiment have shown marked success. Takeshita and co-workers showed that intramuscular administration of VEGF significantly increased capillary density and limb perfusion. 33A significant increase in neovascularisation has also been demonstrated when VEGF was administered by transfecting a VEGF gene-bearing vector directly into the animal? 4,35In addition, infusion of VEGF directly into the vascular pedicle supplying an axial pattern skin flap was also extremely effective.14Evidently then, VEGF is capable of exerting a powerful angiogenic effect irrespective of the method in which it is delivered. In conclusion, the use of growth factors such as VEGF holds promise in improving skin flap viability when distal necrosis is a potential complication of a flap-related procedure. By inducing neovascularisation, thus increasing the blood supply to the flap, VEGF is able to protect the flap from hypoxia and the

British Journal of Plastic Surgery resulting damage. VEGF is effective when delivered in a variety of methods, including subdermal, subfascial and systemic injections, as well as when applied topically to the recipient bed. Future efforts should address dose and route of administration that are most effective in the clinical setting.

Acknowledgements Thanks to Len Newlin for assistance with surgical and laboratory technique and to Dante Campagna-Pinto and David Levin for help with the histological analysis. Also, thanks to Genentech, Inc. for supplying us with the VEGE

References 1. Nail AV, Brownlee RE, Colvin CP, et al. Transforming growth factor beta t improves wound healing and random flap survival in normal and irradiated rats. Arch Otolaryngol Head Neck Surg 1996; 122: 171-7. 2. Horn DB, Baker SR, Graham LM, McClatchey KD. Utilizing angiogenic agents to expedite the neovascularization process in skin flaps. Laryngoscope 1988; 98:521 6. 3. Ishiguro N, Yabe Y, Shimizu T, lwata H, Miura T. Basic fibroblast growth factor has a beneficial effect on the viability of random skin flaps in rats. Ann Plast Surg 1994; 32: 356-60. 4. Horn DB, Assefa G. Effects of endothelial cell growth factor on vascular compromised skin flaps. Arch Otolaryngol Head Neck Surg 1992; 118: 624-8. 5. Pu LQ, Gadowski GR, Graham AM, et al. Enhanced revascularisation after angiogenic stimulation in a rabbit model of bilateral limb ischaemia. Eur J Vasc Endovasc Surg 1995, 9:189-96. 6. Lefer AM, Ma XL, Weyrich AS, Scalia R. Mechanism of the cardioprotective effect of transforming growth factor beta 1 in feline myocardial ischemia and reperfusion. Proc Natl Acad Sci USA 1993; 90: 1018-22. 7. Kenny D, Coughlan MG, Pagel PS, Kampine JP, Warltier DC. Transforming growth factor beta 1 preserves endothelial function after multiple brief coronary artery occlusions and reperfusion. Am Heart J 1994; 127:1456 61. 8. Keck PJ, Hauser SD, Krivi G, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGE Science 1989; 246: 1309-12. 9. Nicosia RF, Lin Y J, Hazelton D, Qian X. Endogenous regulation of angiogenesis in the rat aorta model. Role of vascular endothelial growth factor. Am J Pathol 1997; 151: 1379-86. 10. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxiainitiated angiogenesis. Nature 1992; 359: 843-5. 11. Hashimoto E, Ogita T, Nakaoka T, Matsuoka R, Takao A, Kira Y. Rapid induction of vascular endothelial growth factor expression by transient ischemia in rat heart. Am J Physiol 1994; 267: H1948-54. 12. Lantieri LA, Martin-Garcia N, Wechsler J, Mitrofanoff M, Raulo Y, Baruch JR Vascular endothelial growth factor expression in expanded tissue: a possible mechanism of angiogenesis in tissue expansion. Plast Reconstr Surg 1998; 101: 392-8. 13. Wysocki SJ, Zheng MH, Smith A, Norman PE. Vascular endothelial growth factor (VEGF) expression during arterial repair in the pig. Eur J Vasc Endovasc Surg 1998; 15: 225-30. 14. Padubidri A, Browne E Jr. Effect of vascular endothelial growth factor (VEGF) on survival of random extension of axial pattern skin flaps in the rat. Ann Plast Surg 1996; 37: 604-11. 15. McFarlane RM, DeYoung G, Henry RA. The design of pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965; 35: 177-82. 16. Davies BW, Lewis RD, Pennington G. The impact Of vasodilators on random-pattern skin flap survival in the rat following mainstream smoke exposure. Ann Plast Surg 1998; 40: 6306.

Effects o f V E G F o n r a n d o m flap survival 17. Carmeliet P, Moons L, Dewerchin M, et al. Insights in vessel development and vascular disorders using targeted inactivation and transfer of vascular endothelial growth factor, the tissue factor receptor, and the plasminogen system. Ann N Y Acad Sei 1997; 811: 191-206. 18. Flamme I, yon Reutern M, Drexler HC, Syed-Ali S, Risau W. Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation. Dev Biol 1995; 171: 399414. 19. Hippenstiel S, Krull M, Ikemann A, Risau W, Clauss M, Suttorp N. VEGF induces hyperpermeability by a direct action on endothelial cells. Am J Physiol 1998; 274:L678 84. 20. Wang W, Merrill ML Borchardt RT. Vascular endothelial growth factor affects permeability of brain microvessel endothelial cells in vitro. Am J Physiol 1996; 271:C1973 80. 21. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380:435 9. 22. Li J, Brown LE Hibberd MG, Grossman JD, Morgan JP, Simons M. VEGF, ilk-l, and fit-1 expression in a rat myocardial infarction model of angiogenesis. Am J Physiol 1996; 270: H1803-11. 23. Takeshita S, Rossow ST, Kearney M, et al. Time course of increased cellular proliferation in collateral arteries after administration of vascular endothelial growth factor in a rabbit model of lower limb vascular insufficiency. Am J Pathol 1995; 147:1649 60. 24. Asahara T, Bauters C, Zheng LP, et al. Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in vivo. Circulation 1995; 92:365 71. 25. Bauters C, Asahara T, Zheng LP, et al. Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol 1994; 267:H1263 71. 26. Walder CE, Errett CJ, Bunting S, et al. Vascular endothelial growth factor augments muscle blood flow and function in a rabbit model of chronic hindlimb ischemia. J Cardiovasc Pharmacol 1996; 27:91 8. 27. Ibukiyama C. Angiogenesis. Angiogenic therapy using fibroblast growth factors and vascular endothelial growth factors lbr ischemic vascular lesions. Jpn Heart J 1996; 37: 285-300. 28. Stein 1, Neeman M, Shweiki D, Itin A, Keshet E. Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes. Mol Cell Biol 1995; 15:5363 8. 29. Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth t:actor. J Cell Sci 1995; 108: 2369-79.

239 30. Wang H J, Chen TM, Chow LS, Cheng TY, Chen JL. Recipient bed vascularity and the survival of ischaemic flaps. Br J Plast Surg 1997; 50: 266-71. 31. Bauters C, Asahara T, Zheng LP, et al. Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. J Vase Surg 1995; 21:314-24. 32. Hopkins SP, Bulgrin JP, Sims RL, Bowman B, Donovan DL, Schmidt SP. Controlled delivery of vascular endothelial growth factor promotes neovascularization and maintains limb function in a rabbit model of ischemia. J Vase Surg 1998; 27:886 94. 33. Takeshita S, Pu LQ, Stein LA, et al. Intramuscular administration of vascular endothelial growth factor induces dosedependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation 1994; 90:II228 34. 34. Tsurumi Y, Kearney M, Chert D, et al. Treatment of acute limb ischemia by intramuscular injection of vascular endothelial growth factor gene. Circulation 1997; 96:II382 8. 35. Mack CA, Magovern CJ, Budenbender KT, et al. Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion. J Vase Surg 1998; 27: 699-709.

The A u t h o r s Zol Kryger BS, Research Fellow Division of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford, California, USA.

Feng Zhang MD, PhD, Instructor William C. Lineaweaver MD, Professor Division of Plastic Surgery, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216, USA. Teoman Dogan MD, Research Fellow Chester Cheng MD, Clinical Fellow Harry J. Buneke MD, Professor Microsurgical Replantation Transplantation Department, California Pacific Medical Center, Davies Campus, San Fransisco, California, USA. Correspondence to Dr William C. Lineaweaver. Paper received 3 November 1998. Accepted 15 December 1999, after revision.