Rat Balloon Injury Model Research Articles

Benidipine, a Calcium Channel Blocker, Regulates Proliferation and Phenotype of Vascular Smooth Muscle Cells

Hyperproliferation of phenotypically modified vascular smooth muscle cells (VSMCs) is one of the major factors in the development of atherosclerosis and restenosis. Previously it was demonstrated that benidipine, a dihydropyridine-calcium channel antagonist, reduced neointimal formation in a rat balloon-injury model. In the present study, we examined the effect of benidipine on the phenotypic modulation and proliferation of VSMCs, using primary cultures of rat VSMCs. In the absence of drug treatment, protein levels of the smooth muscle specific markers, such as smooth muscle myosin heavy chain-1 (SM1), calponin 1, and alpha-actin, decreased during culture. However, treatment of VSMCs with benidipine (3 - 10 micromol/L) for 1 week reversed the effect in a concentration-related manner so that high levels of marker proteins were maintained. The expression of calponin mRNAs was reduced markedly during 1-week culture, and treatment with benidipine (3 micromol/L) significantly inhibited the reduction. Treatment with benidipine for 2 days increased the level of p21 protein and partially reduced p70 S6 kinase 1 (p70S6K1) activity. These data suggest that benidipine may arrest the growth of VSMCs, thereby preventing cell dedifferentiation. These additional properties of benidipine suggest that the drug should provide useful therapy for atherosclerosis and restenosis.

Oxidized LDL receptor LOX-1 is involved in neointimal hyperplasia after balloon arterial injury in a rat model

LOX-1 is a multi-ligand receptor originally identified as the endothelial oxidized LDL receptor. LOX-1 expression is also induced in smooth muscle cells in response to proinflammatory and oxidative stimuli. Here, we report on the role of LOX-1 in intimal hyperplasia, in which proinflammatory and oxidative stimuli are increased. Left common carotid artery of rat was injured by a balloon catheter. The expression of LOX-1 was significantly increased within 24 h after the balloon injury and peaked at day 7. LOX-1 expression was observed predominantly in medial smooth muscle cells until day 3, and then shifted to predominantly intimal smooth muscle cells. At day 14, the expression was concentrated in the regenerated endothelial cells. To examine the contributory role of LOX-1 in the growth of intimal smooth muscle cells, rats were administered anti-LOX1 antibody intravenously every 3 days after balloon injury. Anti-LOX-1 antibody administration effectively suppressed intimal hyperplasia, oxidative stress, and leukocyte infiltration compared with control IgG. These findings suggest the importance of LOX-1 expression in the pathogenesis of neointimal formation in conjunction with oxidative stress and leukocyte infiltration. The LOX-1 expressed in smooth muscle cells is involved in intimal hyperplasia in a rat model of balloon injury. Manipulation of LOX-1 activity is a novel potential therapeutic target to prevent restenosis after angioplasty.

Mechanisms of the Inhibitory Effect of Epigallocatechin-3-Gallate on Cultured Human Vascular Smooth Muscle Cell Invasion

Although we recently showed that the administration of catechins reduced the neointimal formation in a rat balloon-injury model, the precise molecular mechanisms are largely unknown. In the present study, we tried to determine these mechanisms using an in vitro SMC invasion system. Boyden chamber assay was used to examine the effect of catechins on the invasive behavior of SMCs. The invasive activity of SMCs through collagen gel was restrained by EGCG in a concentration-dependent manner. The data from gelatin and collagen zymography and Western blot revealed that EGCG blocks the activation of pro-matrix metalloproteinase (MMP)-2 during an invasion assay and in the conditioned medium of cultured SMCs as well as the activities of MMP-2 and membrane type 1-MMP (MT1-MMP) even at 0.1 to 0.3 micromol/L of EGCG. EGCG was found to restrain MT1-MMPcat-dependent pro-MMP-2 activation. EGCG upregulated the expression of tissue inhibitor of MMP-2 (TIMP-2) protein. Reverse zymography showed that the increased TIMP-2 to expression was validated by an increased activity. The data from decreased TIMP-2 activity using its siRNA suggested that upregulation of TIMP-2 expression may be one of the major mechanisms for inhibition of SMC invasion by EGCG. These results indicate that EGCG targets multiple MMP-mediated SMC cellular events and provides a new major mechanism for the SMC invasion through upregulation of TIMP-2 expression to modulate MMP activity.

A Novel αvβ3 Integrin Antagonist Suppresses Neointima Formation for More Than 4 Weeks After Balloon Injury in Rats

We performed a detailed kinetic analysis in a rat balloon injury model to clarify the essential roles of alphavbeta3 integrin and endothelial cell (EC) regeneration in neointima formation. Using this model, we evaluated the antistenotic effect of Dainippon compound BS-1417, a novel alphavbeta3 integrin antagonist. Kinetic analysis using RT-PCR showed that alphavbeta3 integrin-related genes are upregulated before neointima formation. Morphological and functional analyses revealed that EC regeneration requires >4 weeks after injury, and that recovery of EC normal function coincides with the arrest of neointima formation. Subcutaneous infusion of BS-1417 for 2, 4, 7, or 12 weeks after injury potently inhibited neointima formation without affecting EC regeneration. Although withdrawal of treatment with BS-1417 after short-term administration after injury resulted in catch-up growth of neointima, a long-term study suggested that this catch-up growth can be prevented by continuous administration of BS-1417 until EC regeneration. We clarified that alphavbeta3 integrin and EC regeneration play an essential role in neointima formation, and that continuous administration of BS-1417 potently and stably inhibits neointima formation without affecting EC regeneration. These findings suggest that BS-1417 might be useful as a novel systemic drug for the treatment of restenosis.

Nitric oxide-generating hydrogels inhibit neointima formation

This study evaluated the effects of localized delivery of nitric oxide (NO) from hydrogels covalently modified with S-nitrosocysteine (Cys-NO) on neoinitma formation, a key component of restenosis, in a rat balloon-injury model. Soluble Cys-NO was used in preliminary studies to identify dosage ranges that were able to simultaneously inhibit smooth muscle cell proliferation, enhance endothelial cell proliferation, and reduce platelet adhesion. Photo-cross-linked PEG-based hydrogels were formed with covalently immobilized Cys-NO. These materials release NO for approximately 24 h and can be applied to tissues and photo-cross-linked in situ to form local drug-delivery systems. Localized delivery of NO from hydrogels containing Cys-NO inhibited neointima formation in a rat balloon-injury model by approximately 75% at 14 days.

Discovery of a Potent and Selective αvβ3 Integrin Antagonist with Strong Inhibitory Activity Against Neointima Formation in Rat Balloon Injury Model.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Green tea catechins inhibit neointimal hyperplasia in a rat carotid arterial injury model by TIMP-2 overexpression.

Although it has been demonstrated that the antioxidant properties of tea catechins reduce atherosclerotic lesions in various animal models of hyperlipidemia, it is not yet clear whether these catechins prevent hyperlipidemia-independent arterial remodeling induced by balloon angioplasty. We evaluated the influence of the administration of the tea extract on vascular remodeling in a rat carotid artery balloon-injury model. Male Wistar rats were supplied drinking water with or without green tea extract (1 mg/ml) supplement. Administration of the tea extract reduced the area of the intima (30%) and the ratio of the intimal area to the medial area (36.2%) in injured arteries compared with those of control rats at 14 days after the injury. Real-time RT-PCR, Western blot, and gelatin zymography revealed a significant increase in tissue inhibitor of matrix metalloproteinase (MMP)-2 (TIMP-2) expression as well as a significant reduction of gelatinolytic net activity and activated MMP-2 levels in the injured arteries as a result of the administration of the tea extract compared with those of control group. Similarly, epigallocatechin-3-gallate, a major constituent of green tea catechins, significantly upregulated TIMP-2 expression in cultured smooth muscle cells. Immunohistochemical analysis showed that the increase of TIMP-2 protein occurred preferentially in the developing neointima. These results indicate that catechins inhibit intimal hyperplasia in a rat balloon-injury model through the upregulation of TIMP-2 expression to modulate MMP activity.

Discovery of a potent and selective $alpha;v$beta;3 integrin antagonist with strong inhibitory activity against neointima formation in rat balloon injury model

Discovery of a potent and selective $alpha;v$beta;3 integrin antagonist with strong inhibitory activity against neointima formation in rat balloon injury model

Discovery of a potent and selective α vβ 3 integrin antagonist with strong inhibitory activity against neointima formation in rat balloon injury model

A new series of phenylpiperazine-based derivatives with strong antagonistic activity for α vβ 3 integrin were synthesized. Of these derivatives, the fluorine-substituted compound 8 showed strong inhibitory activity and high selectivity for α vβ 3 integrin receptor (IC 50 = 0.055 nM). In vivo evaluation of the antistenotic effects of 8 indicated that this compound significantly inhibits neointima formation in rat balloon injury model.

Neuronal nitric oxide synthase as a novel anti-atherogenic factor.

Nitric oxide (NO) has multiple important actions that contribute to the maintenance of vascular homeostasis. NO is synthesized by three different isoforms of NO synthase (NOS), all of which have been reported to be expressed in human atherosclerotic vascular lesions. Although the regulatory roles of endothelial NOS (eNOS) and inducible NOS (iNOS) on the development of atherosclerosis have been described, little is known about the role of neuronal NOS (nNOS). Recent studies have demonstrated that nNOS also exerts important vasculoprotective effects in vivo. In a carotid artery ligation model, nNOS-knockout mice exhibited accelerated neointimal formation and constrictive vascular remodeling caused by blood flow disruption. In a rat balloon injury model, the selective inhibition of nNOS activity potently enhanced vasoconstrictor responses to a variety of calcium-mobilizing stimuli, and exacerbated neointimal formation. Moreover, in apolipoprotein E-knockout mice, deficiency of nNOS induced progression of aortic vascular lesion formation. In these models, nNOS was up-regulated in vascular lesions, and was predominantly expressed in the neointima and medial smooth muscle cells. These results provide the first direct evidence that nNOS plays important roles in suppressing arteriosclerotic vascular lesion formation. Thus, nNOS could be regarded as a novel anti-atherogenic factor.

Expression of cell cycle regulators during smooth muscle cell proliferation after balloon catheter injury of rat artery.

Intimal hyperplasia is defined as the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) with deposition of extracellular matrix. However, the cell cycle regulatory mechanisms of injury-induced VSMC proliferation are largely unknown. To examine the expression kinetics of cell cycle regulatory factors which is known to be worked positively or negatively, we used rat balloon injury model. Marked induction of proliferating cell nuclear antigen (PCNA), G1/S cyclin-dependent kinase (cdk2), and its regulatory subunit (cyclin E) occurred between 1 and 3 days after balloon arterial injury, and this was sustained for up to 7 days and then declined. However, the induction of the negative regulators, p21 and p27, occurred between 3 and 5 days of injury, peaked after 7 and 14 days and was then sustained. VSMC proliferation after balloon catheter injury of the rat iliac artery is associated with coordinated expression of positive (cdk2, cyclin E and PCNA) and negative (p21, p27) regulators. Cell cycle regulators such as cdk2, cyclin E, p21, p27 may be suitable targets for the control of intimal hyperplasia.

Activation of apoptosis signal-regulating kinase 1 in injured artery and its critical role in neointimal hyperplasia.

Apoptosis signal-regulating kinase 1 (ASK1), recently identified as one of the mitogen-activated protein kinase kinase kinases, is activated by various extracellular stimuli and involved in a variety of cellular function. Therefore, we first examined the role of ASK1 in vascular remodeling. We used rat balloon injury model and cultured vascular smooth muscle cells (VSMCs). Arterial ASK1 activity was rapidly and dramatically increased after balloon injury. To specifically inhibit endogenous ASK1 activation, dominant-negative mutant of ASK1 (DN-ASK1) was transfected into rat carotid artery before balloon injury. Gene transfer of DN-ASK1 significantly prevented neointimal formation at 14 days after injury. Bromodeoxyuridine labeling index at 7 days after injury showed that DN-ASK1 remarkably suppressed VSMC proliferation in both the intima and the media. We also examined the role of ASK1 in cultured rat VSMCs. Infection with DN-ASK1 significantly attenuated serum-induced VSMC proliferation and migration. We also compared neointimal formation after cuff placement around the femoral artery between mice deficient in ASK1 (ASK1-/- mice) and wild-type (WT) mice. Neointimal formation at 28 days after cuff injury in ASK1-/- mice was significantly attenuated compared with WT mice. Furthermore, we compared the proliferation and migration of VSMCs isolated from ASK1-/- mice with WT mice. Both proliferation and migration of VSMCs from ASK1-/- mice were significantly attenuated compared with VSMCs from WT mice. ASK1 activation plays the key role in vascular intimal hyperplasia. ASK1 may provide the basis for the development of new therapeutic strategy for vascular diseases.

4P-1136 Kinetic analysis of the regulatory of endothelial cell regeneration in smooth muscle cell proliferation: Evaluation of endothelial cells function in rat balloon injury model

4P-1136 Kinetic analysis of the regulatory of endothelial cell regeneration in smooth muscle cell proliferation: Evaluation of endothelial cells function in rat balloon injury model

Vasculoprotective roles of neuronal nitric oxide synthase.

Nitric oxide (NO) has multiple important actions that contribute to the maintenance of vascular homeostasis. NO is synthesized by three different isoforms of NO synthase (NOS), all of which have been reported to be expressed in human atherosclerotic vascular lesions. Although the regulatory roles of endothelial NOS (eNOS) and inducible NOS (iNOS) on the development of atherosclerosis have been described, little is known about the role of neuronal NOS (nNOS). Here, we show that nNOS also exerts important vasculoprotective effects in vivo. In a carotid artery ligation model, nNOS gene-deficient (nNOS-KO) mice exhibited accelerated neointimal formation and constrictive vascular remodeling caused by blood flow disruption. In a rat balloon injury model, the selective inhibition of nNOS activity potently enhanced vasoconstrictor responses to a variety of calcium-mobilizing stimuli, suppressed tissue cGMP concentrations, a marker of vascular NO production, and exacerbated neointimal formation. In both models, nNOS was absent before injury and was up-regulated only after the injury, and was predominantly expressed in the neointima and medial smooth muscle cells. These results provide the first direct evidence that nNOS plays important roles in suppressing arteriosclerotic vascular lesion formation in vivo.

Administration of recombinant P-selectin glycoprotein ligand Fc fusion protein suppresses inflammation and neointimal formation in Zucker diabetic rat model.

P-selectin-mediated leukocyte-endothelium and leukocyte-platelet interaction has been reported after vascular injury and has been correlated with neointimal hyperplasia, but its role in neointimal formation after arterial injury in diabetes has not been described. Using a Zucker diabetic rat balloon injury model, we examined the role of P-selectin in the vascular inflammatory process and neointimal formation after balloon injury. Immunohistochemistry revealed that P-selectin was intensely expressed and that CD45-positive leukocyte infiltration was significantly increased after arterial injury. A single preprocedural intravenous administration of a recombinant P-selectin-soluble glycoprotein ligand-Ig inhibited CD45-positive leukocyte accumulation and suppressed neointimal formation in the Zucker diabetic rat model. These results suggest that reduction of P-selectin-mediated leukocyte activation with the use of recombinant P-selectin-soluble glycoprotein ligand-Ig decreases the inflammatory response and limits neointimal formation after balloon injury in diabetes.

Ubiquitin, Proteasome, and Restenosis

In the present issue of Circulation , Meiners and his colleagues1 show that local application of a proteasome inhibitor resulted in significant inhibition of intimal hyperplasia in a balloon injury model in rat carotid artery. This report is of interest because it identifies a novel target for the treatment of restenosis and introduces a rapidly developing field in basic science to cardiovascular researchers. Proteasomes are large intracellular protein complexes that specialize in degrading cellular proteins that are either unneeded or damaged.2–5⇓⇓⇓ Inhibiting the activity of proteasome not only stops the ability of the cell to get rid of unwanted proteins, but also blocks most cellular processes critical for a cell’s survival. Inhibitors of proteasome activity have been used widely in basic research and more recently in anticancer clinical trials. It is not surprising that proteasome inhibitors can inhibit intimal hyperplasia because they generally have antiproliferative, antiinflammatory, and proapoptotic properties. In order to understand the inner workings of proteasome, one must become acquainted with a small protein called ubiquitin that serves as a molecular tag directing unneeded or damaged proteins to the proteasome for degradation. See p 483 Ubiquitin, a 76 amino acid protein, is highly conserved in evolution with only a 3 amino acid difference between yeast and human homologues. Most of the known function of ubiquitin resides in its ability to covalently attach to other proteins through the formation of an isopeptide bond attaching the C-terminal Glycine residue of ubiquitin to a Lysine residue of the target protein.2,4,5⇓⇓ A second ubiquitin then attaches to the first ubiquitin to build up a polyubiquitin chain, which is required for the tagged protein to be recognized by the proteasome. The process of attaching ubiquitin molecules to other proteins is termed ubiquitination; it occurs through …

Smoothelin is an indicator of reversible phenotype modulation of smooth muscle cells in balloon-injured rat carotid arteries.

Restenosis is the major obstacle interfering with a successful long-term outcome of balloon angioplasty. Neointima formation following endothelial injury is the result of phenotype modulation and proliferation of smooth muscle cells (SMC). To characterize these time-dependent changes, a rat balloon injury model of carotid artery restenosis was assessed. We applied monoclonal antibodies recognizing desmin, sm-alpha-actin and smoothelin, a novel marker specific for the differentiated phenotype of SMC. Neointima formation could be seen from day 7 after injury onwards. During early phases, the number of smoothelin-positive cells in the media was decreased compared with uninjured controls. Smoothelin staining was absent in the neointima during formation. Increased levels of smoothelin in both media and neointima were observed at days 28 and 56, correlating with a decrease in proliferation as assessed by Ki-67 antigen staining. No such changes were observed for desmin and sm-alpha-actin. Following balloon injury, SMC in both the media and the neointima underwent an early, reversible dedifferentiation, followed by proliferation. The novel SMC-specific marker protein smoothelin can be used to monitor this SMC (de)differentiation in neointima and media. These findings support the pivotal role of SMC phenotype modulation in neointima formation and restenosis.

Effects of probucol on rat carotid artery responses to balloon catheter injury

Balloon catheter injury to the rat common carotid artery has been widely used for testing potential therapies for post-angioplasty restenosis. However, the model has become somewhat discredited because a number of drugs that inhibit intimal thickening, measured 14 days after balloon catheter injury, have been found to be ineffective in clinical trials. Probucol has recently been shown to reduce the incidence of post-angioplasty restenosis in a number of small clinical trials, making it possible to reassess the validity of the rat balloon injury model. The effects of probucol on the underlying causes of intimal thickening in balloon-injured rat carotid arteries were quantified. Probucol inhibited medial smooth muscle cell proliferation by 23% on day 4 after injury ( P=0.009), and by 65% on day 10 after injury ( P=0.026). Smooth muscle cell migration was reduced by 64% ( P=0.008) in probucol-treated animals. In marked contrast, intimal smooth muscle cell proliferation was significantly increased by 41% ( P=0.024) by probucol. There was no significant effect on intimal thickening or smooth muscle cell death. These data suggest that drugs that inhibit both medial smooth muscle cell proliferation and migration in the rat balloon injury model may prove useful in the treatment of post-angioplasty restenosis.

Roles of P-selectin in inflammation, neointimal formation, and vascular remodeling in balloon-injured rat carotid arteries.

P-selectin plays key roles in mediating inflammation through promoting adherence of leukocytes to activated platelets and endothelium. This process is one of the initial events in atherosclerosis and restenosis after coronary angioplasty. Using a rat balloon-injury model, we examined the role of P-selectin in vascular inflammatory processes. In the acute phase, immunohistochemistry revealed that P-selectin was intensely expressed on both activated platelets covering the denuded segment and endothelial cells of the inflamed adventitial small vessels. Treatment with an anti-P-selectin monoclonal antibody (MAb) for 8 consecutive days significantly inhibited neointimal formation at day 14 (42% inhibition; P:<0.05), and this effect persisted at day 56 (40% inhibition; P:<0.01) compared with the control group. Vascular shrinking accompanying adventitial fibrosis was also attenuated at day 56. Inhibition of both neointimal formation and vascular shrinking resulted in the lumen area of the anti-P-selectin treatment group being approximately 3 times larger at day 56 than that of the control group. Accumulation of CD45-positive leukocytes in the developing neointima, media, and adventitia at day 8 was significantly inhibited by treatment with the anti-P-selectin MAb. Scanning electron microscopy demonstrated that anti-P-selectin treatment resulted in a less thrombogenic surface of the arterial intima, which featured a pseudoendothelial appearance at day 14 after injury. These results suggest that inhibition of P-selectin-mediated leukocyte recruitment prevents the development of neointimal formation, adventitial inflammation, and vascular shrinking and promotes pseudoendothelialization by luminal smooth muscle cells. This treatment thus beneficially affects vascular remodeling after balloon injury in rats.

In vivo transfer of human hepatocyte growth factor gene accelerates re-endothelialization and inhibits neointimal formation after balloon injury in rat model.

Although most therapeutic strategies to prevent restenosis are designed to inhibit vascular smooth muscle cell (VSMC) proliferation directly, VSMC proliferation might be indirectly inhibited by re-endothelialization, as endothelial cells secrete antiproliferative and antithrombotic substances. We hypothesized that application of an endothelium-specific growth factor to balloon-injured arteries could accelerate re-endothelialization, thereby attenuating intimal hyperplasia. In this study, we investigated in vivo gene transfer of human HGF that exclusively stimulated endothelial cells without replication of VSMC growth into injured vessels. Transfection of human HGF gene into rat balloon-injured carotid artery resulted in significant inhibition of neointimal formation up to at least 8 weeks after transfection, accompanied by detection of human immunoreactive HGF. Induction of re-endothelialization induced by overexpression of human HGF gene transfer into balloon-injured vessels is supported by several lines of evidence: (1) Administration of HGF vector. but not control vector, markedly inhibited neointimal formation, accompanied by a significant increase in vascular human and rat HGF concentrations. (2) Planimetric analysis demonstrated a significant increase in re-endothelialized area in arteries transfected with human HGF vector. (3) Induction of NO content in balloon-injured vessels transfected with human HGF vector was observed in accordance with the recovery of endothelial vasodilator properties in response to acetylcholine. As endogenous HGF expression in balloon-injured vessels was significantly decreased as compared with normal vessels, the present study demonstrated the successful inhibition of neointimal formation by transfection of human HGF gene as 'cytokine supplement therapy' in a rat balloon injury model.