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Neointimal hyperplasia and vascular endothelial growth factor expression are increased in normoglycemic, insulin resistant, obese fatty rats
Atherosclerosis 184 (2006) 283–289
Neointimal hyperplasia and vascular endothelial growth factor expression are increased in normoglycemic, insulin resistant, obese fatty rats Cyrus V. Desouza ∗ , Moira Gerety, Frederick G. Hamel Research Service, Omaha Veterans Affairs Medical Center, Section of Diabetes, Endocrinology and Metabolism, University of Nebraska Medical Center, Omaha, NE, USA Received 31 January 2005; received in revised form 18 April 2005; accepted 27 April 2005 Available online 6 June 2005
Abstract Objective: Insulin resistance is associated with a constellation of factors that enhance the artherosclerotic process. Vessel injury results in the formation of a markedly increased neointima in type 2 diabetes. Increased neointimal hyperplasia (NH) and vascular endothelial growth factor (VEGF) expression may lead to restenosis post angioplasty. We studied NH and VEGF expression in an obese, insulin resistant, but normoglycemic rat model, after carotid balloon injury. Methods and results: Diabetic rats (ZDF, n = 10), normoglycemic, insulin-resistant rats (ZDF-normoglycemic, n = 6) as well as Zucker fatty rats (FZ, n = 6), and lean Zucker rats (LZ, n = 6), all 13–16 weeks old, were subjected to right carotid injury by an angioplasty catheter introduced via the femoral artery. Three weeks later the rats were sacrificed and serum and carotids obtained. The intima–media ratio (I/M) was then calculated. ZDF-normoglycemic, FZ and ZDF-diabetic rats were all hyperinsulinemic and hypertriglyceridemic when compared to LZ rats. ZDF diabetic rats were hyperglycemic while FZ, ZDF-normoglycemic and LZ rats were normoglycemic. The I/M ratio for ZDF and FZ rats were significantly greater than for LZ rats. The VEGF expression was significantly greater in ZDF and FZ rats than LZ rats. Conclusions: In conclusion, insulin resistance increases neointimal hyperplasia and VEGF expression even with normoglycemia, after carotid angioplasty in rats. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neointimal hyperplasia; Insulin resistance; Hyperinsulinemia; Vascular endothelial growth factor; Type 2 diabetes; Vascular injury
1. Introduction Over 24% of the adult population in the United States, has insulin resistance without overt diabetes [1]. Insulin resistance is associated with a constellation of factors that enhance the artherosclerotic process [2–6]. Hyperinsulinemia, which may represent compensation for insulin resistance (IR), may further accelerate vascular injury [2,7–9]. Restenosis is a common and serious complication following angioplasty and stent implantation in patients with arterial vascular disease. Consequent to vessel injury, there is an ∗ Corresponding author at: Omaha Veterans Affairs Medical Center, Medicine Department (111), 4101 Woolworth Avenue, Omaha, NE 68105, USA. Tel.: +1 402 346 8800x5506; fax: +1 402 977 5602. E-mail address: [email protected] (C.V. Desouza).
0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.04.015
inflammatory response, which results in the formation of a neointima [10,11]. Left unabated, inflammation leads to neointimal hyperplasia and restenosis, which is markedly increased in type 2 diabetes animal models when compared to normal and type 1 diabetes models [12,13]. Neointimal formation in an insulin resistant model without the additional factor of hyperglycemia has not been studied. As the number of individuals who have insulin resistance exceeds those with type 2 diabetes, understanding the role of insulin resistance in endothelial injury and regrowth is important. Vascular endothelial growth factor (VEGF) expression in the vasculature of diabetes and insulin resistant animal models differ. Studies have shown that there is decreased expression of VEGF in the macrovasculature and increased expression in the microvasculature [14]. Some studies show that VEGF mediated stimulation of intimal hyperplasia,
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following mechanical injury to the vessel wall after angioplasty, may play a role in restenosis [15,16]. VEGF may also contribute to plaque instability [17]. However, other studies show a reduction of neointimal hyperplasia and accelerated re-endothelialization with VEGF treatment [18]. Thus, the role of VEGF on neointimal hyperplasia is not clear. Moreover its role in intimal hyperplasia post angioplasty in type 2 diabetes and insulin resistant normoglycemic models has not been studied. Thus, we investigated the expression of VEGF and its association with intimal hyperplasia in insulin resistant normoglycemic rats.
2. Methods 2.1. Animal models The ZDF Gmi fa/fa inbred obese female rat (Charles River Lab.) is a non-diabetic model of insulin resistance when on a normal diet. It is hyperinsulinemic, insulin resistant, normoglycemic and normotensive. It has mild to moderate hypertriglyceridemia. However, when the female rat is fed a diet of RD 13004 (Charles River Lab.), it develops diabetes in 1 month and is then a good model of type 2 diabetes. Both non-diabetic and diabetic models were used. The male rat develops diabetes on a normal diet and hence was not used. The Zucker fa/fa obese non-diabetic female rat (Charles River Lab.) is a non-diabetic model of insulin resistance. It has marked obesity, hyperinsulinemia and insulin resistance. It has mild hypertriglyceridemia. However, it is normotensive and normoglycemic. The lean Zucker female rat has normal metabolic parameters. 2.2. Study design Four groups of rats were used. Groups consisted of Zucker fatty non-diabetic female rats (n = 6), ZDF non-diabetic female rats (n = 6), ZDF diabetic female rats (n = 10) and lean Zucker female rats (n = 6). Rats were subjected to carotid angioplasty as described below. The final number of rats that survived till the time of euthanasia was Zucker fatty non-diabetic (n = 4), ZDF non-diabetic (n = 6), ZDF diabetic (n = 9), Zucker lean (n = 6). All procedures and protocols were approved by the Subcommittee of Animal Studies and the Research and Development Committee of the Omaha VAMC. 2.3. Angioplasty Zucker fa/fa obese rats, ZDF non-diabetic rats, ZDF diabetic rats and lean Zucker rats, (age, 13 weeks) were used. Angioplasty was done using a modified version of a method that has been used by us before [10,13]. Briefly rats were anesthetized with isoflourane and the inner left thigh was shaved and cleaned. Under aseptic conditions, an incision
was made to expose the left femoral artery. Hemostasis was achieved with an arterial clamp. A 2.3 F, 2 mm, balloon length 20 mm, percutaneous transluminal coronary angioplasty (PTCA) catheter (Boston SCI-Med) was introduced into the femoral artery. The catheter was directed to the left carotid artery, with the help of a guide wire, under fluoroscopic guidance. Angioplasty was done at 4 atm. Then the pressure was reduced to 2 atm and the balloon dragged down the entire carotid, from its bifurcation to the aortic arch as done in previous experiments. The balloon was then deflated and removed. The femoral artery was ligated and hemostasis achieved. The wound was closed in three layers. Skin staples were used. Rats were under direct close observation for the next 30 min. All procedures were done under a dissecting microscope and in the setting of a sterile vivarial operating room. Using this procedure, neointimal thickness is detectable within 1 week and is extensive by 3 weeks. Rats were euthanized with carbon dioxide. Carotid arteries were perfused with formalin prior to removal. Through a midline cervical incision, both the left and the right carotid, from the aortic arch to the bifurcation were removed and preserved in formalin. 2.4. Morphometric analysis Morphometric analysis was done using a previously described method [13,19,20]. Briefly the left carotid from the junction of the aortic arch to the junction of the bifurcation was taken. Formaldehyde-fixed carotids were embedded in paraffin. The carotid was sectioned into four equal parts. Four sections from the two middle parts at 3 mm intervals were used for analysis. The neoinitimal hyperplasia is maximal in the middle of the vessel as endothelial regrowth occurs from the aortic root and from the bifurcation and is last to regrow in this area. All four sections as described above were selected for measurements. Sections were stained with hematoxylin. Analysis on the stained sections was done with 10× microscopic magnifications. Computerized digital microscopic software (ANA@lysis) was used to obtain measurements of the intimal and medial areas by a previously described method [13,19]. The intima/media ratios were then obtained. 2.5. Metabolic parameters Blood was obtained at the time of euthanasia. Serum insulin was measured by radioimmunoassay using rat specific anti-insulin antibodies (Linco Labs, St. Louis, MO). Serum triglycerides were measured using the triglyceride slide method (Johnson and Johnson, Rochester, NY). Blood glucose was monitored daily from 1 week prior to angioplasty, to the day of euthanasia. A mean blood glucose level was then calculated using all the values. A Freestyle (Therasense) glucose monitor was used to measure blood glucose. This was done as there is a fluctuation of blood sugar levels due to the stress of surgery and a one-time laboratory
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blood glucose level would not reflect that. Moreover, amounts of blood needed to do laboratory blood glucose levels daily, exceeded that safely allowed for a rat. Hence a glucometer was used. 2.6. VEGF measurements Immunohistochemistry was done using previously described methods [21–23]. Cross-sections of formalinfixed, paraffin-embedded tissue were used for analysis. The cross-sections were obtained as described earlier in the morphometric analysis section. Each sample was deparaffinized in xylene and gradually rehydrated in ethanol. After blocking endogenous peroxidase with 30% hydrogen peroxide in methanol for 20 min, an antigen retrieval step was added by microwaving the samples in citrate buffer (pH 6.0) for 30 min. The samples were then washed three times with PBS (phosphate buffered saline), followed by incubation with Chem Mate blocking antibody solution (DAKO, Carpinteria, CA) for 30 min. The samples were then incubated for 1 h at room temperature with murine anti-VEGF (Oncogene, San Diego, CA), diluted 1:50 in PBS. The samples were then washed and incubated with ready to use DAKO EnVision+, Peroxidase, for 30 min at room temperature. After incubation, the slides were rinsed in PBS and then exposed to stable diaminobenzidine tetrahydrochloride (DAB) and the reaction monitored under light microscopy. The sections were then counterstained with Mayer’s hematoxylin. 2.6.1. Quantification Four sections from each rat were analyzed. Sections were obtained as described earlier under morphometric analysis. Tissue images were recorded using a SPOT cooled color digital camera, linked to a computer. Digitized software (AN@lysis) was used to measure the staining intensity of the neointima. The total intensity of staining of all the pixels in the neointimal area were measured and then divided by the total number of pixels in the neointima, to obtain a mean staining density value. 2.7. Statistical analysis All statistical analysis was performed with Sigma Stat software (SSPS, Chicago, IL). One way ANOVA was performed on the data with pair-wise multiple comparison procedures (Tukey test) as a post-test. Data are reported as
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Table 2 Intimal hyperplasia and VEGF expression 3 weeks after angioplasty Rat species
Intima/media ratio
Intimal VEGF staining intensity
Lean Zucker Zucker non-diabetic ZDF non-diabetic ZDF diabetic
0.67 ± 0.06 1.28 ± 0.12* 1.4 ± 0.10* 1.6 ± 0.12*
46 ± 7.3 84 ± 6.2* 94 ± 4.3* 98 ± 7*
*
P < 0.01 vs. Lean Zucker.
mean ± S.E.M. Statistical significance was set at an α of <0.05. 3. Experimental results 3.1. Metabolic parameters Lean Zucker, Zucker non-diabetic and ZDF non-diabetic rats had normal blood glucose levels and were not significantly different from each other (Table 1). ZDF diabetic rats had elevated blood glucose levels consistent with diabetes when compared to the other rat groups. Zucker non-diabetic, ZDF non-diabetic and ZDF diabetic rats all had significantly elevated fasting plasma insulin levels when compared to lean Zucker rats. ZDF diabetic rats had a trend towards higher insulin levels than Zucker non-diabetic and ZDF non-diabetic rats. However, this did not reach statistical significance. Body weight at sacrifice was significantly greater in Zucker nondiabetic, ZDF non-diabetic and ZDF diabetic rats when compared to lean Zucker rats, but not significantly different from each other. Triglycerides were elevated in all rat groups when compared to the lean Zucker group. There was no significant difference between the other groups, although there was a trend toward lower triglycerides in the Zucker non-diabetic rats when compared to the ZDF rats (Table 1). 3.2. Intima–media ratio The intima–media ratio was determined 3 weeks after carotid angioplasty as described in the methods section. The intima–media ratio for Zucker non-diabetic rats (1.28 ± 0.12), ZDF non-diabetic (1.4 ± 0.10) and ZDF diabetic rats (1.6 ± 0.12), were all significantly greater than the lean Zucker rats (0.67 ± 0.06)(Table 2 and Fig. 1). Pair wise testing showed that the intima–media ratio between the insulin resistant non-diabetic groups and insulin-resistant diabetic group were not significantly different.
Table 1 Metabolic parameters at euthanasia (3 weeks post-angioplasty) Rat species
Glucose (mg/dl)
Lean Zucker Zucker non-diabetic ZDF non-diabetic ZDF diabetic
84 83 91.6 377
*
P < 0.01 vs. Lean Zucker.
± ± ± ±
2 5 7 27*
Insulin (ng/ml)
Body weight (gm)
Triglycerides (mg/dl)
0.73 ± 0.14 5.3 ± 0.8* 4.4 ± 0.76* 6.3 ± 0.58*
221 ± 13 451 ± 32* 397 ± 24* 412 ± 28*
90.2 532 806 744
± ± ± ±
11 30* 32* 44*
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Fig. 1. Neointimal hyperplasia post carotid angioplasty in: (A) Lean Zucker, (B) ZDF non-diabetic, and (C) ZDF diabetic rats.
Fig. 2. VEGF expression in: (A) Lean Zucker, (B) ZDF non-diabetic, and (C) ZDF diabetic rats.
3.3. VEGF protein expression VEGF protein was measured at 3 weeks post-angioplasty as described in the methods section. The intimal VEGF protein staining intensity was significantly greater in Zucker non-diabetic rats, ZDF non-diabetic and ZDF diabetic rats when compared to Lean Zucker rats (Table 2 and Fig. 2). Pair wise testing showed that there was no significant difference in VEGF protein expression between the insulin resistant nondiabetic and insulin resistant diabetic rats.
4. Discussion Insulin resistance is associated with a constellation of factors that enhance the artherosclerotic process [2–6]. Hyperinsulinemia, which may represent compensation for insulin resistance (IR), may further accelerate vascular injury [2,7–9]. Neointimal hyperplasia is greater in patients with diabetes after coronary interventions, which often leads to restenosis after angioplasty [24–26]. A study by Park et al., [12] demonstrated that neointimal hyperplasia in the obese ZDF rat (type 2 diabetic rat model), was twice as much as the lean Zucker controls and streptozotocin-treated Sprague-Dawley rats (type 1 diabetic
model) [12]. The difference between the type 1 and type 2 diabetes models was hyperinsulinemia in the type 2 model. However, both models also had hyperglycemia. Several studies have shown that hyperglycemia itself may not be the key factor in inducing neointimal hyperlasia, post angioplasty. Type 1 diabetic, hyperglycemic insulin-deficient rats show decreased or similar degree of neointimal hyperplasia as normal controls [12,19,27]. When these rats are then given enough insulin to control glucose levels, neointimal hyperplasia actually increased [27]. However, all these studies had the additional factor of hyperglycemia and hence a diabetic milieu, which adds a number of confounding factors. To our knowledge no studies evaluating neointimal hyperplasia in the pre-diabetic phase of insulin resistance, where hyperglycemia is absent, but hyperinsulinemia is present, have been reported. In our study, we used various insulin resistant rat models to try and mimic the spectrum of disease seen in humans, from normal to insulin resistance to diabetes. The lean rats had much less neointimal hyperplasia than the insulin resistant models. However, the insulin resistant normoglycemic rats had as much neointimal hyperplasia as the insulin resistant diabetic rats. Insulin levels between diabetic and non-diabetic hyperinsulinemic rats were elevated, but not significantly different. Yet with the additional factor of hyperglycemia the degree of neointimal hyperplasia did not change. Thus from
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our study it is reasonable to conclude that hyperinsulinemia or the insulin resistant state may be more important than hyperglycemia in neointima formation and post-angioplasty re-stenosis, in these rat models. However, this is an association between hyperinsulinemia and neointimal hyperplasia and needs to be further clarified in future studies. The effects of VEGF on the vasculature are controversial. In the choroidal and retinal circulation of the eye, VEGF exerts proliferative and inflammatory responses, which may contribute to the worsening of diabetic retinopathy [28–31]. Other studies have shown that VEGF may induce vascular smooth muscle cell activation and cytokine production post injury [32,33]. However, VEGF is a powerful mitogen for endothelial cells and has been shown to hasten endothelial cell regrowth. Several studies show that treatment with VEGF can accelerate re-endothelialization and attenuate intimal hyperplasia post balloon injury [34–37]. In our study, VEGF expression was greatly increased in the insulin resistant normoglycemic as well as diabetic rats, when compared to the lean rats. As with the neointimal hyperplasia the VEGF expression was increased to the same extent in the diabetic, hyperinsulinemic rat, as the normoglycemic, hyperinsulinemic rats. There was a similar association between VEGF and hyperinsulinemia as the association between neointimal hyperplasia and hyperinsulinemia. Thus, in our study it would seem that VEGF might have deleterious effects on vascular healing post-angioplasty. In vitro studies in vascular smooth muscle cells and endothelial cells have shown that insulin stimulates VEGF expression [38,39]. Aortic smooth muscle cells obtained from insulin receptor substrate-1 knockout (IRS-1)−/−) mice showed a marked decrease in VEGF expression in response to insulin stimulation [40]. As VEGF is a growth factor, it may increase the size of the neointimal proliferation in the setting of hyperinsulinemia and insulin resistance. The role of the renin–angiotensin system (RAS) in vascular pathology has been extensively studied. The RAS system is also implicated in insulin resistance and the metabolic syndrome [41,42]. The RAS system is upregulated after tissue injury, which results in detrimental effects on inflammation, cell growth, proliferation and vascular function [43,44]. Angiotensin II has been shown to potentiate VEGF induced proliferation, which could encourage neointimal hyperplasia [45,46]. Animal studies have shown that angiotensin converting enzyme (ACE) inhibition as well as well as angiotensin receptor blockade, lead to decreased neointimal hyperplasia in rats after balloon injury [47]. Moreover, several human studies have shown that ACE inhibitors may reduce insulin resistance [48]. Thus, it is possible that part of the vascular pathology associated with the RAS is due to its interactions with VEGF and insulin sensitivity. Further studies are required to clarify the underlying mechanisms. Nitric oxide (NO) plays a vital role in vascular function and pathology. Several studies have shown that NO release is important for the function of the intact endothelium [49–51]. However, NO produced by inducible nitric oxide synthase
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(iNOS) is proinflammatory [51]. VEGF stimulates release of NO in VSMC and endothelial cells [52]. Studies have also shown that NO in turn up-regulates VEGF expression and secretion [53]. This may set up a local paracrine effect where NO and VEGF increase each other’s expression and thereby induce VSMC proliferation [53]. Our study shows increased VEGF expression after angioplasty. Hence, interactions between VEGF and NO may have contributed to the increased neointimal hyperplasia seen in our study. Thus, we conclude that hyperinsulinemia or the insulin resistant state, even in the absence of hyperglycemia, was associated with an increase in neointimal hyperplasia and VEGF expression. Further studies are required to elucidate the role of VEGF on the vasculature in a hyperinsulinemic state.
Acknowledgement This work was supported by a grant(s) from the Department of Veterans Affairs Merit Review Entry Program (CVD, ID 00195).
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Neointimal hyperplasia and vascular endothelial growth factor expression are increased in normoglycemic, insulin resistant, obese fatty rats Cyrus V. Desouza ∗ , Moira Gerety, Frederick G. Hamel Research Service, Omaha Veterans Affairs Medical Center, Section of Diabetes, Endocrinology and Metabolism, University of Nebraska Medical Center, Omaha, NE, USA Received 31 January 2005; received in revised form 18 April 2005; accepted 27 April 2005 Available online 6 June 2005
Abstract Objective: Insulin resistance is associated with a constellation of factors that enhance the artherosclerotic process. Vessel injury results in the formation of a markedly increased neointima in type 2 diabetes. Increased neointimal hyperplasia (NH) and vascular endothelial growth factor (VEGF) expression may lead to restenosis post angioplasty. We studied NH and VEGF expression in an obese, insulin resistant, but normoglycemic rat model, after carotid balloon injury. Methods and results: Diabetic rats (ZDF, n = 10), normoglycemic, insulin-resistant rats (ZDF-normoglycemic, n = 6) as well as Zucker fatty rats (FZ, n = 6), and lean Zucker rats (LZ, n = 6), all 13–16 weeks old, were subjected to right carotid injury by an angioplasty catheter introduced via the femoral artery. Three weeks later the rats were sacrificed and serum and carotids obtained. The intima–media ratio (I/M) was then calculated. ZDF-normoglycemic, FZ and ZDF-diabetic rats were all hyperinsulinemic and hypertriglyceridemic when compared to LZ rats. ZDF diabetic rats were hyperglycemic while FZ, ZDF-normoglycemic and LZ rats were normoglycemic. The I/M ratio for ZDF and FZ rats were significantly greater than for LZ rats. The VEGF expression was significantly greater in ZDF and FZ rats than LZ rats. Conclusions: In conclusion, insulin resistance increases neointimal hyperplasia and VEGF expression even with normoglycemia, after carotid angioplasty in rats. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neointimal hyperplasia; Insulin resistance; Hyperinsulinemia; Vascular endothelial growth factor; Type 2 diabetes; Vascular injury
1. Introduction Over 24% of the adult population in the United States, has insulin resistance without overt diabetes [1]. Insulin resistance is associated with a constellation of factors that enhance the artherosclerotic process [2–6]. Hyperinsulinemia, which may represent compensation for insulin resistance (IR), may further accelerate vascular injury [2,7–9]. Restenosis is a common and serious complication following angioplasty and stent implantation in patients with arterial vascular disease. Consequent to vessel injury, there is an ∗ Corresponding author at: Omaha Veterans Affairs Medical Center, Medicine Department (111), 4101 Woolworth Avenue, Omaha, NE 68105, USA. Tel.: +1 402 346 8800x5506; fax: +1 402 977 5602. E-mail address: [email protected] (C.V. Desouza).
0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.04.015
inflammatory response, which results in the formation of a neointima [10,11]. Left unabated, inflammation leads to neointimal hyperplasia and restenosis, which is markedly increased in type 2 diabetes animal models when compared to normal and type 1 diabetes models [12,13]. Neointimal formation in an insulin resistant model without the additional factor of hyperglycemia has not been studied. As the number of individuals who have insulin resistance exceeds those with type 2 diabetes, understanding the role of insulin resistance in endothelial injury and regrowth is important. Vascular endothelial growth factor (VEGF) expression in the vasculature of diabetes and insulin resistant animal models differ. Studies have shown that there is decreased expression of VEGF in the macrovasculature and increased expression in the microvasculature [14]. Some studies show that VEGF mediated stimulation of intimal hyperplasia,
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following mechanical injury to the vessel wall after angioplasty, may play a role in restenosis [15,16]. VEGF may also contribute to plaque instability [17]. However, other studies show a reduction of neointimal hyperplasia and accelerated re-endothelialization with VEGF treatment [18]. Thus, the role of VEGF on neointimal hyperplasia is not clear. Moreover its role in intimal hyperplasia post angioplasty in type 2 diabetes and insulin resistant normoglycemic models has not been studied. Thus, we investigated the expression of VEGF and its association with intimal hyperplasia in insulin resistant normoglycemic rats.
2. Methods 2.1. Animal models The ZDF Gmi fa/fa inbred obese female rat (Charles River Lab.) is a non-diabetic model of insulin resistance when on a normal diet. It is hyperinsulinemic, insulin resistant, normoglycemic and normotensive. It has mild to moderate hypertriglyceridemia. However, when the female rat is fed a diet of RD 13004 (Charles River Lab.), it develops diabetes in 1 month and is then a good model of type 2 diabetes. Both non-diabetic and diabetic models were used. The male rat develops diabetes on a normal diet and hence was not used. The Zucker fa/fa obese non-diabetic female rat (Charles River Lab.) is a non-diabetic model of insulin resistance. It has marked obesity, hyperinsulinemia and insulin resistance. It has mild hypertriglyceridemia. However, it is normotensive and normoglycemic. The lean Zucker female rat has normal metabolic parameters. 2.2. Study design Four groups of rats were used. Groups consisted of Zucker fatty non-diabetic female rats (n = 6), ZDF non-diabetic female rats (n = 6), ZDF diabetic female rats (n = 10) and lean Zucker female rats (n = 6). Rats were subjected to carotid angioplasty as described below. The final number of rats that survived till the time of euthanasia was Zucker fatty non-diabetic (n = 4), ZDF non-diabetic (n = 6), ZDF diabetic (n = 9), Zucker lean (n = 6). All procedures and protocols were approved by the Subcommittee of Animal Studies and the Research and Development Committee of the Omaha VAMC. 2.3. Angioplasty Zucker fa/fa obese rats, ZDF non-diabetic rats, ZDF diabetic rats and lean Zucker rats, (age, 13 weeks) were used. Angioplasty was done using a modified version of a method that has been used by us before [10,13]. Briefly rats were anesthetized with isoflourane and the inner left thigh was shaved and cleaned. Under aseptic conditions, an incision
was made to expose the left femoral artery. Hemostasis was achieved with an arterial clamp. A 2.3 F, 2 mm, balloon length 20 mm, percutaneous transluminal coronary angioplasty (PTCA) catheter (Boston SCI-Med) was introduced into the femoral artery. The catheter was directed to the left carotid artery, with the help of a guide wire, under fluoroscopic guidance. Angioplasty was done at 4 atm. Then the pressure was reduced to 2 atm and the balloon dragged down the entire carotid, from its bifurcation to the aortic arch as done in previous experiments. The balloon was then deflated and removed. The femoral artery was ligated and hemostasis achieved. The wound was closed in three layers. Skin staples were used. Rats were under direct close observation for the next 30 min. All procedures were done under a dissecting microscope and in the setting of a sterile vivarial operating room. Using this procedure, neointimal thickness is detectable within 1 week and is extensive by 3 weeks. Rats were euthanized with carbon dioxide. Carotid arteries were perfused with formalin prior to removal. Through a midline cervical incision, both the left and the right carotid, from the aortic arch to the bifurcation were removed and preserved in formalin. 2.4. Morphometric analysis Morphometric analysis was done using a previously described method [13,19,20]. Briefly the left carotid from the junction of the aortic arch to the junction of the bifurcation was taken. Formaldehyde-fixed carotids were embedded in paraffin. The carotid was sectioned into four equal parts. Four sections from the two middle parts at 3 mm intervals were used for analysis. The neoinitimal hyperplasia is maximal in the middle of the vessel as endothelial regrowth occurs from the aortic root and from the bifurcation and is last to regrow in this area. All four sections as described above were selected for measurements. Sections were stained with hematoxylin. Analysis on the stained sections was done with 10× microscopic magnifications. Computerized digital microscopic software (ANA@lysis) was used to obtain measurements of the intimal and medial areas by a previously described method [13,19]. The intima/media ratios were then obtained. 2.5. Metabolic parameters Blood was obtained at the time of euthanasia. Serum insulin was measured by radioimmunoassay using rat specific anti-insulin antibodies (Linco Labs, St. Louis, MO). Serum triglycerides were measured using the triglyceride slide method (Johnson and Johnson, Rochester, NY). Blood glucose was monitored daily from 1 week prior to angioplasty, to the day of euthanasia. A mean blood glucose level was then calculated using all the values. A Freestyle (Therasense) glucose monitor was used to measure blood glucose. This was done as there is a fluctuation of blood sugar levels due to the stress of surgery and a one-time laboratory
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blood glucose level would not reflect that. Moreover, amounts of blood needed to do laboratory blood glucose levels daily, exceeded that safely allowed for a rat. Hence a glucometer was used. 2.6. VEGF measurements Immunohistochemistry was done using previously described methods [21–23]. Cross-sections of formalinfixed, paraffin-embedded tissue were used for analysis. The cross-sections were obtained as described earlier in the morphometric analysis section. Each sample was deparaffinized in xylene and gradually rehydrated in ethanol. After blocking endogenous peroxidase with 30% hydrogen peroxide in methanol for 20 min, an antigen retrieval step was added by microwaving the samples in citrate buffer (pH 6.0) for 30 min. The samples were then washed three times with PBS (phosphate buffered saline), followed by incubation with Chem Mate blocking antibody solution (DAKO, Carpinteria, CA) for 30 min. The samples were then incubated for 1 h at room temperature with murine anti-VEGF (Oncogene, San Diego, CA), diluted 1:50 in PBS. The samples were then washed and incubated with ready to use DAKO EnVision+, Peroxidase, for 30 min at room temperature. After incubation, the slides were rinsed in PBS and then exposed to stable diaminobenzidine tetrahydrochloride (DAB) and the reaction monitored under light microscopy. The sections were then counterstained with Mayer’s hematoxylin. 2.6.1. Quantification Four sections from each rat were analyzed. Sections were obtained as described earlier under morphometric analysis. Tissue images were recorded using a SPOT cooled color digital camera, linked to a computer. Digitized software (AN@lysis) was used to measure the staining intensity of the neointima. The total intensity of staining of all the pixels in the neointimal area were measured and then divided by the total number of pixels in the neointima, to obtain a mean staining density value. 2.7. Statistical analysis All statistical analysis was performed with Sigma Stat software (SSPS, Chicago, IL). One way ANOVA was performed on the data with pair-wise multiple comparison procedures (Tukey test) as a post-test. Data are reported as
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Table 2 Intimal hyperplasia and VEGF expression 3 weeks after angioplasty Rat species
Intima/media ratio
Intimal VEGF staining intensity
Lean Zucker Zucker non-diabetic ZDF non-diabetic ZDF diabetic
0.67 ± 0.06 1.28 ± 0.12* 1.4 ± 0.10* 1.6 ± 0.12*
46 ± 7.3 84 ± 6.2* 94 ± 4.3* 98 ± 7*
*
P < 0.01 vs. Lean Zucker.
mean ± S.E.M. Statistical significance was set at an α of <0.05. 3. Experimental results 3.1. Metabolic parameters Lean Zucker, Zucker non-diabetic and ZDF non-diabetic rats had normal blood glucose levels and were not significantly different from each other (Table 1). ZDF diabetic rats had elevated blood glucose levels consistent with diabetes when compared to the other rat groups. Zucker non-diabetic, ZDF non-diabetic and ZDF diabetic rats all had significantly elevated fasting plasma insulin levels when compared to lean Zucker rats. ZDF diabetic rats had a trend towards higher insulin levels than Zucker non-diabetic and ZDF non-diabetic rats. However, this did not reach statistical significance. Body weight at sacrifice was significantly greater in Zucker nondiabetic, ZDF non-diabetic and ZDF diabetic rats when compared to lean Zucker rats, but not significantly different from each other. Triglycerides were elevated in all rat groups when compared to the lean Zucker group. There was no significant difference between the other groups, although there was a trend toward lower triglycerides in the Zucker non-diabetic rats when compared to the ZDF rats (Table 1). 3.2. Intima–media ratio The intima–media ratio was determined 3 weeks after carotid angioplasty as described in the methods section. The intima–media ratio for Zucker non-diabetic rats (1.28 ± 0.12), ZDF non-diabetic (1.4 ± 0.10) and ZDF diabetic rats (1.6 ± 0.12), were all significantly greater than the lean Zucker rats (0.67 ± 0.06)(Table 2 and Fig. 1). Pair wise testing showed that the intima–media ratio between the insulin resistant non-diabetic groups and insulin-resistant diabetic group were not significantly different.
Table 1 Metabolic parameters at euthanasia (3 weeks post-angioplasty) Rat species
Glucose (mg/dl)
Lean Zucker Zucker non-diabetic ZDF non-diabetic ZDF diabetic
84 83 91.6 377
*
P < 0.01 vs. Lean Zucker.
± ± ± ±
2 5 7 27*
Insulin (ng/ml)
Body weight (gm)
Triglycerides (mg/dl)
0.73 ± 0.14 5.3 ± 0.8* 4.4 ± 0.76* 6.3 ± 0.58*
221 ± 13 451 ± 32* 397 ± 24* 412 ± 28*
90.2 532 806 744
± ± ± ±
11 30* 32* 44*
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Fig. 1. Neointimal hyperplasia post carotid angioplasty in: (A) Lean Zucker, (B) ZDF non-diabetic, and (C) ZDF diabetic rats.
Fig. 2. VEGF expression in: (A) Lean Zucker, (B) ZDF non-diabetic, and (C) ZDF diabetic rats.
3.3. VEGF protein expression VEGF protein was measured at 3 weeks post-angioplasty as described in the methods section. The intimal VEGF protein staining intensity was significantly greater in Zucker non-diabetic rats, ZDF non-diabetic and ZDF diabetic rats when compared to Lean Zucker rats (Table 2 and Fig. 2). Pair wise testing showed that there was no significant difference in VEGF protein expression between the insulin resistant nondiabetic and insulin resistant diabetic rats.
4. Discussion Insulin resistance is associated with a constellation of factors that enhance the artherosclerotic process [2–6]. Hyperinsulinemia, which may represent compensation for insulin resistance (IR), may further accelerate vascular injury [2,7–9]. Neointimal hyperplasia is greater in patients with diabetes after coronary interventions, which often leads to restenosis after angioplasty [24–26]. A study by Park et al., [12] demonstrated that neointimal hyperplasia in the obese ZDF rat (type 2 diabetic rat model), was twice as much as the lean Zucker controls and streptozotocin-treated Sprague-Dawley rats (type 1 diabetic
model) [12]. The difference between the type 1 and type 2 diabetes models was hyperinsulinemia in the type 2 model. However, both models also had hyperglycemia. Several studies have shown that hyperglycemia itself may not be the key factor in inducing neointimal hyperlasia, post angioplasty. Type 1 diabetic, hyperglycemic insulin-deficient rats show decreased or similar degree of neointimal hyperplasia as normal controls [12,19,27]. When these rats are then given enough insulin to control glucose levels, neointimal hyperplasia actually increased [27]. However, all these studies had the additional factor of hyperglycemia and hence a diabetic milieu, which adds a number of confounding factors. To our knowledge no studies evaluating neointimal hyperplasia in the pre-diabetic phase of insulin resistance, where hyperglycemia is absent, but hyperinsulinemia is present, have been reported. In our study, we used various insulin resistant rat models to try and mimic the spectrum of disease seen in humans, from normal to insulin resistance to diabetes. The lean rats had much less neointimal hyperplasia than the insulin resistant models. However, the insulin resistant normoglycemic rats had as much neointimal hyperplasia as the insulin resistant diabetic rats. Insulin levels between diabetic and non-diabetic hyperinsulinemic rats were elevated, but not significantly different. Yet with the additional factor of hyperglycemia the degree of neointimal hyperplasia did not change. Thus from
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our study it is reasonable to conclude that hyperinsulinemia or the insulin resistant state may be more important than hyperglycemia in neointima formation and post-angioplasty re-stenosis, in these rat models. However, this is an association between hyperinsulinemia and neointimal hyperplasia and needs to be further clarified in future studies. The effects of VEGF on the vasculature are controversial. In the choroidal and retinal circulation of the eye, VEGF exerts proliferative and inflammatory responses, which may contribute to the worsening of diabetic retinopathy [28–31]. Other studies have shown that VEGF may induce vascular smooth muscle cell activation and cytokine production post injury [32,33]. However, VEGF is a powerful mitogen for endothelial cells and has been shown to hasten endothelial cell regrowth. Several studies show that treatment with VEGF can accelerate re-endothelialization and attenuate intimal hyperplasia post balloon injury [34–37]. In our study, VEGF expression was greatly increased in the insulin resistant normoglycemic as well as diabetic rats, when compared to the lean rats. As with the neointimal hyperplasia the VEGF expression was increased to the same extent in the diabetic, hyperinsulinemic rat, as the normoglycemic, hyperinsulinemic rats. There was a similar association between VEGF and hyperinsulinemia as the association between neointimal hyperplasia and hyperinsulinemia. Thus, in our study it would seem that VEGF might have deleterious effects on vascular healing post-angioplasty. In vitro studies in vascular smooth muscle cells and endothelial cells have shown that insulin stimulates VEGF expression [38,39]. Aortic smooth muscle cells obtained from insulin receptor substrate-1 knockout (IRS-1)−/−) mice showed a marked decrease in VEGF expression in response to insulin stimulation [40]. As VEGF is a growth factor, it may increase the size of the neointimal proliferation in the setting of hyperinsulinemia and insulin resistance. The role of the renin–angiotensin system (RAS) in vascular pathology has been extensively studied. The RAS system is also implicated in insulin resistance and the metabolic syndrome [41,42]. The RAS system is upregulated after tissue injury, which results in detrimental effects on inflammation, cell growth, proliferation and vascular function [43,44]. Angiotensin II has been shown to potentiate VEGF induced proliferation, which could encourage neointimal hyperplasia [45,46]. Animal studies have shown that angiotensin converting enzyme (ACE) inhibition as well as well as angiotensin receptor blockade, lead to decreased neointimal hyperplasia in rats after balloon injury [47]. Moreover, several human studies have shown that ACE inhibitors may reduce insulin resistance [48]. Thus, it is possible that part of the vascular pathology associated with the RAS is due to its interactions with VEGF and insulin sensitivity. Further studies are required to clarify the underlying mechanisms. Nitric oxide (NO) plays a vital role in vascular function and pathology. Several studies have shown that NO release is important for the function of the intact endothelium [49–51]. However, NO produced by inducible nitric oxide synthase
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(iNOS) is proinflammatory [51]. VEGF stimulates release of NO in VSMC and endothelial cells [52]. Studies have also shown that NO in turn up-regulates VEGF expression and secretion [53]. This may set up a local paracrine effect where NO and VEGF increase each other’s expression and thereby induce VSMC proliferation [53]. Our study shows increased VEGF expression after angioplasty. Hence, interactions between VEGF and NO may have contributed to the increased neointimal hyperplasia seen in our study. Thus, we conclude that hyperinsulinemia or the insulin resistant state, even in the absence of hyperglycemia, was associated with an increase in neointimal hyperplasia and VEGF expression. Further studies are required to elucidate the role of VEGF on the vasculature in a hyperinsulinemic state.
Acknowledgement This work was supported by a grant(s) from the Department of Veterans Affairs Merit Review Entry Program (CVD, ID 00195).
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