Drug-coated balloon therapy for in-stent restenosis in patients with iliofemoral deep vein thrombosis: A single-arm observational study.

BACKGROUND: Iliofemoral deep vein thrombosis (IFDVT) causes severe symptoms and affect the quality of life to a great extent. Endovascular thrombectomy and stent implantation have been a feasible strategie to alleviate the signs and symptoms of IFDVT. However, venous in-stent restenosis (ISR) has become an emerging non-negligible problem. METHODS: To evaluate the histological characteristics of venous ISR, neointima of arterial and venous ISR patients were collected and examed. To explore the effect of drug-coated balloon (DCB) on venous ISR lesions, we conducted a single-center retrospective case series study involving IFDVT patients with ISR after venous stenting who were treated with paclitaxel-coated balloon dilatation. RESULTS: We found a collagen-rich matrix but not elastin, as well as fewer cells and less neovascularization in venous intimal hyperplasia compared with neointima in arteries. Thirteen IFDVT patients were involved in the study, with average preoperative stenosis degree of 87.69% ± 13.48%. After intervention, the stenosis degree was significantly reduced to 14.6% ± 14.36% immediately (p < 0.0001) and to 16.54% ± 15.73% during follow-up (p < 0.0001). During follow-up, the VEINES-QOL scores (p < 0.0001), VEINES-Sym scores (p < 0.0001), and Villalta scores (p = 0.04) of patients was improved significantly compared with those before intervention. No major adverse events were observed. CONCLUSIONS: The use of DCB may have a positive effect in the treatment of venous ISR by targeting intimal hyperplasia. Moreover, the application of DCB dilatation in IFDVT stenting patients with ISR is deemed safe and effective.

Sulfamethizole attenuates poloxamer 407-induced atherosclerotic neointima formation via inhibition of mTOR in C57BL/6 mice.

Mammalian target of Rapamycin C1 (mTORC1) inhibition limits plaque progression in atherosclerosis. The present study evaluated the protective effect of sulfamethizole on poloxamer 407-induced atherosclerotic neointima formation in C57BL/6 mice via mTOR inhibition. Poloxamer 407 (P-407) (0.5 g/kg body weight) was administered intraperitoneally to male C57BL/6 mice every third day for 148 days to induce chronic hyperlipidemia. From Day 121 to 148, animals were additionally administered Sulfamethizole (5, 10, and 50 mg/kg, p.o.), Rapamycin (0.5 mg/kg, positive control), or vehicle (1 ml/kg). Plasma lipid levels were measured on Days 120 and 148. Upon sacrifice, histological studies were performed, and aortic tissue interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and mTOR levels were evaluated. A molecular docking study was carried out to mimic the interaction of sulfamethizole with mTOR protein. Chronic P-407 administration significantly (p < 0.001) elevated plasma lipid levels, compared with those of the normal control group. Chronic hyperlipidemia resulted in increased tunica intima thickness, collagen deposition, and IL-6, TNF-α, and mTOR levels. Treatment with Sulfamethizole attenuated these parameters significantly in a dose-dependent manner. Molecular docking studies showed a significant interaction of Sulfamethizole with mTOR. In conclusion, this study suggests that sulfamethizole significantly limits poloxamer 407-induced atherosclerotic neointima formation in C57BL/6 mice via mTOR inhibition.

Activation of TFEB ameliorates dedifferentiation of arterial smooth muscle cells and neointima formation in mice with high-fat diet.

Autophagy is recently implicated in regulating vascular smooth muscle cell (SMC) homeostasis and in the pathogenesis of vascular remodeling. Transcription factor EB (TFEB) is a master regulator of autophagy signaling pathways. However, the molecular mechanisms and functional roles of TFEB in SMC homeostasis have not been elucidated. Here, we surveyed the ability of TFEB to regulate autophagy pathway in SMCs, and whether pharmacological activation of TFEB favors SMC homeostasis preventing dedifferentiation and pathogenic vascular remodeling. In primary cultured SMCs, TFEB activator trehalose induced nuclear translocation of TFEB and upregulation of TFEB-controlled autophagy genes leading to enhanced autophagy signaling. Moreover, trehalose suppressed serum-induced SMC dedifferentiation to synthetic phenotypes as characterized by inhibited proliferation and migration. These effects of trehalose were mimicked by ectopic upregulation of TFEB and inhibited by TFEB gene silencing. In animal experiments, partial ligation of carotid arteries induced downregulation of TFEB pathway in the media layer of these arteries. Such TFEB suppression was correlated with increased SMC dedifferentiation and aggravated high-fat diet (HFD)-induced neointima formation. Treatment of mice with trehalose reversed this TFEB pathway suppression, and prevented SMC dedifferentiation and HFD-induced neointima formation. In conclusion, our findings have identified TFEB as a novel positive regulator for autophagy pathway and cellular homeostasis in SMCs. Our data suggest that suppression of TFEB may be an initiating mechanism that promotes SMC dedifferentiation leading to accelerated neointima formation in vascular disorders associated with metabolic stress, whereas trehalose reverses these changes. These findings warrant further evaluation of trehalose in the clinical settings.

2,3,7,8-Tetrachlorodibenzo-p-dioxin promotes injury-induced vascular neointima formation in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant that causes cardiovascular toxicity. The phenotypic transformation of vascular smooth muscle cells (VSMCs) from the contractile to the synthetic phenotype is a hallmark of vascular response to injury. However, the precise role and molecular mechanism of TCDD in vascular remodeling remains unknown. In the present study, we found that TCDD treatment promoted VSMC phenotypic transition from contractile to synthetic phenotype and exaggerated vascular neointimal hyperplasia after wire injury in mice. TCDD treatment enhanced VSMC entry into cell cycle from G0/G1 phase to S and G2/M phase. The expression of cyclin D1, cyclin-dependent kinase 4 (CDK4), and its phosphorylation were coordinately increased in response to TCDD treatment. Knocking down of aryl hydrocarbon receptor (AHR) inhibited VSMC phenotypic transition induced by TCDD and promoted S/G2 phase cell cycle arrest. TCDD treatment markedly increased oncogenic c-Jun gene expression in VSMCs. ChIP assay revealed the direct binding of AHR on the promoter of c-Jun to up-regulate the mRNA expression of c-Jun. Silencing of c-Jun gene enhanced the expression of p53 and p21, whereas attenuated the expression of CDK4 and cyclin D1 leading to the decrease in the TCDD-stimulated VSMC proliferation and synthetic phenotype transition in vitro. In vivo study showed that genetic ablation of c-Jun in VSMCs restricted injury-induced neointimal hyperplasia in TCDD-treated mice. Thus, TCDD exposure exaggerated injury-induced vascular remodeling by the activation of AHR and up-regulation of the expression of its target gene c-Jun, indicating that inhibition of AHR may be a promising prevention strategy for TCDD-associated cardiovascular diseases.-Guo, S., Zhang, R., Liu, Q., Wan, Q., Wang, Y., Yu, Y., Liu, G., Shen, Y., Yu, Y., Zhang, J. 2,3,7,8-Tetrachlorodibenzo-p-dioxin promotes injury-induced vascular neointima formation in mice.

Xanthohumol Blocks Proliferation and Migration of Vascular Smooth Muscle Cells in Vitro and Reduces Neointima Formation in Vivo.

Xanthohumol (1) is a principal prenylated chalcone found in hops. The aim of this study was to examine its influence on platelet-derived growth factor (PDGF)-BB-triggered vascular smooth muscle cell (VSMC) proliferation and migration in vitro and on experimentally induced neointima formation in vivo. Quantification of resazurin conversion indicated that 1 can inhibit PDGF-BB-induced VSMC proliferation concentration-dependently (IC50 = 3.49 µM). Furthermore, in a wound-healing assay 1 potently suppresses PDGF-BB-induced VSMC migration at 15 µM. Tested in a mouse femoral artery cuff model, 1 significantly reduces neointima formation. Taken together, we show that 1 represses PDGF-BB-induced VSMC proliferation and migration in vitro as well as neointima formation in vivo. This novel activity suggests 1 as an interesting candidate for further studies addressing a possible therapeutic application to counteract vascular proliferative disease.

Lysophosphatidic acid-induced vascular neointimal formation in mouse carotid arteries is mediated by the matricellular protein CCN1/Cyr61.

Vascular smooth muscle cell (SMC) migration is an essential step involved in neointimal formation in restenosis and atherosclerosis. Lysophosphatidic acid (LPA) is a bioactive component of oxidized low-density lipoprotein and is produced by activated platelets, implying that LPA influences vascular remodeling. Our previous study revealed that matricellular protein CCN1, a prominent extracellular matrix (ECM) protein, mediates LPA-induced SMC migration in vitro. Here we examined the role of CCN1 in LPA-induced neointimal formation. By using LPA infusion of carotid artery in a mouse model, we demonstrated that LPA highly induced CCN1 expression (approximately six- to sevenfold) in neointimal lesions. Downregulation of CCN1 expression with the specific CCN1 siRNA in carotid arteries blocked LPA-induced neointimal formation, indicating that CCN1 is essential in LPA-induced neointimal formation. We then used LPA receptor knockout (LPA1-/-, LPA2-/-, and LPA3-/-) mice to examine LPA receptor function in CCN1 expression in vivo and in LPA-induced neointimal formation. Our data reveal that LPA1 deficiency, but not LPA2 or LPA3 deficiency, prevents LPA-induced CCN1 expression in vivo in mouse carotid arteries. We also observed that LPA1 deficiency blunted LPA infusion-induced neointimal formation, indicating that LPA1 is the major mediator for LPA-induced vascular remodeling. Our in vivo model of LPA-induced neointimal formation established a key role of the ECM protein CCN1 in mediating LPA-induced neointimal formation. Our data support the notion that the LPA1-CCN1 axis may be the central control for SMC migration and vascular remodeling. CCN1 may serve as an important vascular disease marker and potential target for vascular therapeutic intervention.

Increase in Vascular Injury of Sodium Overloaded Mice May be Related to Vascular Angiotensin Modulation.

This study aimed to analyzing the effect of chronic sodium overload upon carotid and femoral injury, and its relation to vascular angiotensin modulation. Male C57Bl6 mice were divided in: control (cont), receiving 1% NaCl solution for 2 weeks (salt-2) or 12 weeks (salt-12). Two-weeks before the end of the study, a 2mm catheter was implanted around the left femoral and carotid arteries to induce injury. Blood pressure (BP) and heart rate (HR) were measured at the end of the study by tail plethysmography. Arteries were collected and prepared for histological analysis to determine arterial thickening and perivascular collagen deposition. Angiotensin II and Ang(1-7) were quantified in fresh arteries using the HPLC method. There were no differences in body weight, BP and HR. Intima/media ratio had a similar increase in both injured arteries of cont and salt-2 mice, but a more pronounced increase was observed in salt-12 mice (31.1±6%). On the other hand, sodium overload modified perivascular collagen deposition, increasing thick fibers (cont: 0.5%; salt-2: 3.4%; salt-12: 0.6%) and decreasing thin fibers (cont: 7.4%; salt-2: 0.5%; salt-12: 6.8%) in non-injured arteries. Injured arteries presented similar collagen fiber distribution. Angiotensin quantification showed increased Ang(1-7) in salt treated mice (salt-2: +72%; salt-12: +45%) with a concomitant decrease in Ang II (salt-2: -54%; salt-12: -60%). Vascular injury increased significantly Ang(1-7) in salt-12 mice (+80%), maintaining Ang II reduction similar to that of a non-injured artery. The lack of changes in BP and HR suggests that the structural changes observed may be due to non-hemodynamic mechanisms such as local renin-angiotensin system. Collagen evaluation suggests that sodium overload induces time-related changes in vascular remodeling. The increase of artery injury with concomitant increase in Ang(1-7) in 12-week treated mice shows a direct association between the duration of salt treatment and the magnitude of vascular injury.

Repetitive hypoglycemia increases circulating adrenaline level with resultant worsening of intimal thickening after vascular injury in male Goto-Kakizaki rat carotid artery.

Hypoglycemia associated with diabetes management is a potential risk for cardiovascular diseases. However, the effect of hypoglycemic episodes including a surge of sympathetic activity on the progression of neointima formation after vascular injury remains largely unknown. In this study, insulin was injected intraperitoneally into nonobese diabetic Goto-Kakizaki (GK) rats, once every 3 days for 4 weeks after balloon injury of carotid artery to induce hypoglycemia. Then, we evaluated balloon injury-induced neointima formation. Insulin treatment enhanced neointima formation and increased the number of proliferating cell nuclear antigen (PCNA)-positive cells in the carotid artery. Injection of glucose with insulin prevented hypoglycemia and abrogated intimal thickening. Also, bunazosin, an α1 adrenergic receptor antagonist, prevented intimal thickening and accumulation of PCNA-positive cells induced by insulin treatment despite the presence of concomitant hypoglycemia and high adrenaline levels. Incubation of cultured smooth muscle cells with adrenaline resulted in a significant increase in their proliferation and G0/G1 to S phase progression, which was associated with activation of extracellular signal-regulated kinase, enhanced expression of cell cycle regulatory molecules such as cyclin D1, and cyclin E, and phosphorylation of retinoblastoma protein. These adrenaline-induced effects were abrogated by bunazosin. Our data indicated that increased adrenaline induced by repetitive hypoglycemia promotes intimal thickening and smooth muscle cell proliferation after endothelial denudation in GK rats.

Endothelial fate mapping in mice with pulmonary hypertension.

BACKGROUND: Pulmonary endothelial injury triggers a reparative program, which in susceptible individuals is characterized by neointima formation, vascular narrowing, and the development of pulmonary arterial hypertension. The neointimal cells in human pathological plexiform lesions frequently coexpress smooth muscle α-actin and the endothelial von Willebrand antigen, creating a question about their cellular lineage of origin. METHODS AND RESULTS: Experimental pulmonary hypertension with neointima formation develops in C57Bl/6 mice subjected to left pneumonectomy followed 1 week later by jugular vein injection of monocrotaline pyrrole (20 µg/µL and 1 µL/g; group P/MCTP). Compared with the group vehicle, by day 35, group P/MCTP developed higher right ventricular systolic pressure (54±5 versus 25±2 mm Hg; P<0.01) and right ventricular hypertrophy (0.58±0.16 versus 0.26±0.05; P<0.01). Transgenic vascular endothelial-cadherin Cre recombinase or Tie-2 Cre mice were intercrossed with mTomato/mGreen fluorescent protein double-fluorescent Cre reporter mice to achieve endothelial genetic lineage marking with membrane-targeted green fluorescent protein. In control mice, few endothelial lineage-marked cells lining the lumen of small pulmonary arteries demonstrate expression of smooth muscle α-actin. Concurrent with the development of pulmonary hypertension, endothelial lineage-marked cells are prominent in the neointima and exhibit expression of smooth muscle α-actin and smooth muscle myosin heavy chain. Human pulmonary arterial hypertension neointimal lesions contain cells that coexpress endothelial CD31 or von Willebrand antigen and smooth muscle α-actin. CONCLUSION: Neointimal cells in pulmonary hypertension include contributions from the endothelial genetic lineage with induced expression of smooth muscle α-actin and smooth muscle myosin heavy chain.

Saturated fatty acid palmitate aggravates neointima formation by promoting smooth muscle phenotypic modulation.

OBJECTIVE: Obesity is a major risk factor of atherosclerotic cardiovascular disease. Circulating free fatty acid levels are known to be elevated in obese individuals and, along with dietary saturated fatty acids, are known to associate with cardiovascular events. However, little is known about the molecular mechanisms by which free fatty acids are linked to cardiovascular disease. APPROACH AND RESULTS: We found that administration of palmitate, a major saturated free fatty acid, to mice markedly aggravated neointima formation induced by carotid artery ligation and that the neointima primarily consisted of phenotypically modulated smooth muscle cells (SMCs). In cultured SMCs, palmitate-induced phenotypic modulation was characterized by downregulation of SMC differentiation markers, such as SM α-actin and SM-myosin heavy chain, and upregulation of mediators involved in inflammation and remodeling of the vessel wall, such as platelet-derived growth factor B and matrix metalloproteinases. We also found that palmitate induced the expression of proinflammatory genes via a novel toll-like receptor 4/myeloid differentiation primary response 88/nuclear factor-κB/NADPH oxidase 1/reactive oxygen species signaling pathway: nuclear factor-κB was activated by palmitate via toll-like receptor 4 and its adapter, MyD88, and once active, it transactivated Nox1, encoding NADPH oxidase 1, a major reactive oxygen species generator in SMCs. Pharmacological inhibition and small interfering RNA-mediated knockdown of the components of this signaling pathway mitigated the palmitate-induced upregulation of proinflammatory genes. More importantly, Myd88 knockout mice were resistant to palmitate-induced exacerbation of neointima formation. CONCLUSIONS: Palmitate seems to promote neointima formation by inducing inflammatory phenotypes in SMCs.

Cariporide, a specific Na/H(+) exchanger 1 blocker, inhibits neointimal proliferation induced by advanced glycation end products in a balloon injury rat model.

AIMS: The association between diabetes and neointimal expansion after vascular injury has been attributed to the accumulation of advanced glycation end products (AGEs). Here we investigated the inhibitory effect of cariporide, a specific Na(+)/H(+) exchanger 1 blocker, on neointimal proliferation induced by AGEs in a balloon injury model. METHODS: Expression of cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP) was monitored by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. The level of reactive oxygen species (ROS) was determined by specific fluorescent probe. The phosphorylation of the nuclear factor-ĸB (NF-ĸB) system was studied by Western blot. RESULTS: Cariporide significantly suppressed AGE-induced neointimal hyperplasia, vascular smooth muscle cell (VSMC) proliferation, COX-2, MMP-2 and MMP-9 expression. In addition, cariporide decreased AGE-induced ROS, malondiadehyde level and increased the superoxide dismutase and glutathione peroxidase activity. We also found that cariporide blocked AGE-induced NF-ĸB activation and inhibitor-ĸB degradation. CONCLUSIONS: The results indicated that cariporide inhibited AGE-induced neointimal formation by suppressing the VSMC proliferation and the up-regulation of COX-2, MMP-2, MMP-9 via inhibiting ROS and NF-ĸB activation.

Plasticity-related gene-1 inhibits lysophosphatidic acid-induced vascular smooth muscle cell migration and proliferation and prevents neointima formation.

Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with ß-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.

Urinary trypsin inhibitor reduced inflammatory response after stent injury in minipig.

This study investigated whether urinary trypsin inhibitor (UTI) inhibits neointimal formation by reducing inflammatory response after stent injury. Twenty minipigs having undergone oversized bare material stent implantation in the left anterior descending artery were randomly subdivided into two groups: a UTI group (n=10) and a control group (n=10). Two systemic markers of inflammation (serum macrophage chemoattractant protein-1 and interleukin-6 levels measured by ELISA) were increased after stent implantation, and two days after stem implantation, their levels were positively correlated with the maximal percentage of area stenosis on day 28 (r(2)=0.889 and 0.743, respectively). This effect was abolished by UTI administration. Twenty-eight days after implantation, morphometric analysis of the stented arteries revealed significantly reduced luminal stenosis (38±6% vs. 64±12%, P<0.05), a neointimal area (3.22±0.57 mm(2) vs. 5.21±1.04 mm(2), P<0.05), neointimal thickness (0.31±0.13 mm vs. 0.46±0.16 mm, P<0.05), and an inflammatory score of 1.02±0.05 vs. 1.30±0.08 in UTI-treated animals as compared with controls. Twenty-eight days after stenting, arterial nuclear factor-κB expression was 36.93±7.16% in all of the cells in controls and 23.32±4.54% in UTI-treated minipigs. UTI could reduce neointimal formation after stenting by inhibiting the local and the systemic inflammatory response. Percutaneous coronary intervention could benefit from precocious anti-inflammatory treatment.

The role of estrogen receptor α and ß in regulating vascular smooth muscle cell proliferation is based on sex.

BACKGROUND: We previously demonstrated that vascular smooth muscle cells (VSMC) proliferation and development of neointimal hyperplasia as well as the ability of nitric oxide (NO) to inhibit these processes is dependent on sex and hormone status. The aim of this study was to evaluate the role of estrogen receptor (ER) in mediating proliferation in male and female VSMC. MATERIALS AND METHODS: Proliferation was assessed in primary rat aortic male and female VSMC using (3)H-thymidine incorporation in the presence or absence of ER alpha (α) inhibitor methyl-piperidino-pyrazole, the ER beta (ß) inhibitor (R,R)-5,11-Diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol, the combined ERαß inhibitor ICI 182,780, and/or the NO donor DETA/NO. Proliferation was also assessed in primary aortic mouse VSMC harvested from wildtype (WT), ERα knockout (ERα KO), and ERß knockout (ERß KO) mice in the presence or absence of DETA/NO and the ERα, ERß, and ERαß inhibitors. Protein levels were assessed using Western blot analysis. RESULTS: Protein expression of ERα and ERß was present and equal in male and female VSMC, and did not change after exposure to NO. Inhibition of either ERα or ERß had no effect on VSMC proliferation in the presence or absence of NO in either sex. However, inhibition of ERαß in rat VSMC mitigated NO-mediated inhibition in female but not male VSMC (P < 0.05). Evaluation of proliferation in the knockout mice revealed distinct patterns. Male ERαKO and ERßKO VSMC proliferated faster than male WT VSMC (P < 0.05). Female ERßKO proliferated faster than female WT VSMC (P < 0.05), but female ERαKO VSMC proliferated slower than female WT VSMC (P < 0.05). Last, we evaluated the effect of combined inhibition of ERα and ERß in these knockout strains. Combined ERαß inhibition abrogated NO-mediated inhibition of VSMC proliferation in female WT and knockout VSMC (P < 0.05), but not in male VSMC. CONCLUSIONS: These data clearly demonstrate a role for the ER in mediating VSMC proliferation in both sexes. However, these data suggest that the antiproliferative effects of NO may be regulated by the ER in females but not males.

Adventitial gene transfer of VEGFR-2 specific VEGF-E chimera induces MCP-1 expression in vascular smooth muscle cells and enhances neointimal formation.

BACKGROUND: The role of vascular endothelial growth factors (VEGFs) in neointimal formation has been controversial. VEGF receptor (R)-2 signaling pathway is crucial in bringing about the effects of VEGFs including vasodilatation, endothelial cell migration and proliferation. In this study we have used an established adventitial gene transfer technique, in vitro studies and a novel VEGF-E/PlGF chimera that binds specifically to VEGFR-2, to investigate the role of VEGFR-2 in neointimal formation. METHODS: Intimal hyperplasia was induced in the carotid arteries of cholesterol fed male New Zealand White rabbits using a silastic collar. Adenoviral vectors encoding VEGF-E chimera (1×10(9) pfu/ml) were transferred to the adventitia of the carotid arteries either alone or together with adenoviruses encoding soluble VEGFR-2 (sVEGFR-2). Adenoviruses encoding LacZ were used as controls. All animals were sacrificed 7 days after the gene transfer. RESULTS: Significant increases in neointimal formation, proliferating cells, inflammatory responses and adventitial angiogenesis were observed in the VEGF-E chimera transduced arteries. The number of medial smooth muscle cells expressing VEGFR-2 was significantly (p<0.001) higher. MCP-1 mRNA levels were significantly (p<0.01) increased in the VEGF-E chimera transduced arteries and transduced rabbit aortic smooth muscle cells (p<0.05). Soluble VEGFR-2 (sVEGFR-2) significantly inhibited VEGF-E chimera induced neointimal formation (p<0.01), cellular proliferation (p<0.01), inflammatory responses (p<0.01) and adventitial angiogenesis (p<0.01). CONCLUSIONS: The results indicate that VEGFR-2 mediated signaling could aggravate neointimal formation and suggest a potential therapeutic role of sVEGFR-2 in inhibiting neointimal formation and adventitial angiogenesis.

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells.

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [(3)H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.

Vascular smooth muscle Jak2 deletion prevents angiotensin II-mediated neointima formation following injury in mice.

The in vitro treatment of vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) causes Janus kinase 2 (Jak2) to interact with the Ang II type 1 receptor (AT(1)-R) resulting in enhanced cell growth. However, the role that Jak2 plays in AT(1)-R-mediated vascular cell growth and remodeling in vivo is less clear. We hypothesized that in vivo, Jak2 plays a rate-limiting role in Ang II-mediated neointima formation following vascular injury. Using the Cre-loxP system, we conditionally ablated Jak2 from the VSMC of mice. We found that these mice are protected from Ang II-mediated neointima formation following iron chloride-induced vascular injury. In addition, the VSMC Jak2 null mice were protected from injury-induced vascular fibrosis and the pathological loss of the contractile marker, smooth muscle α-actin. Finally, when compared to controls, the VSMC Jak2 null mice exhibited significantly less Ang II-induced VSMC proliferation and migration in vitro and in vivo and more apoptosis. These results suggest that Jak2 plays a central role in the causation of Ang II-induced neointima formation following vascular injury and may provide a novel target for the prevention of neointima formation.

Antiretrovirals induce endothelial dysfunction via an oxidant-dependent pathway and promote neointimal hyperplasia.

Human immunodeficiency virus-1 antiretroviral treatment is associated with an increased incidence of atherosclerosis. We hypothesized that antiretrovirals directly impair endothelial function after short-term exposure and that with chronic exposure, this dysfunction promotes a proliferative response, inducing neointimal hyperplasia, thus contributing to vascular lesion formation. To test this hypothesis, we treated mice with the nucleoside reverse transcriptase inhibitor azidothymidine (AZT), the protease inhibitor indinavir, or AZT + indinavir. Treatment with AZT or AZT + indinavir for 5 days impaired endothelium-dependent vessel relaxation. Though indinavir treatment alone did not alter vessel relaxation, it potentiated the impairment of endothelium-dependent relaxation induced by AZT. Coadministration of the antioxidant Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin attenuated antiretroviral-induced endothelial dysfunction, suggesting that oxidant production may have a causal role in the observed endothelial dysfunction. To test whether the antiretrovirals promote a proliferative response following endothelial dysfunction, we treated mice with antiretrovirals for 14 days and then induced a carotid endothelial injury. Two weeks later, we observed a dramatic increase in neointimal formation in all antiretroviral-treated animals, and the newly formed neointima was comprised mainly of proliferated smooth muscle cells. Although a functional endothelium surrounding the lesioned area and re-endothelialization across the area of injury is important in reducing proliferation in this model, we tested whether the neointimal hyperplasia was associated with endothelial dysfunction. Plasma levels of asymmetric dimethylarginine, a biomarker of endothelial dysfunction, increased after treatment with indinavir or AZT + indinavir. On the other hand, treatment with AZT or AZT + indinavir increased endothelial vascular cell adhesion molecule staining. We conclude that short-term treatment with antiretrovirals elicited a direct impairment in endothelial function, in part via an oxidant-dependent pathway. These antiretrovirals also exacerbated injury-induced vascular smooth muscle cell proliferation and neointimal hyperplasia, likely because of their inhibition of endothelial function.