MDM2 dual-color in situ hybridization (DISH) aids the diagnosis of intimal sarcomas.

Intimal sarcoma is a rare malignant mesenchymal tumor arising from the intima of the great vessels and the heart, and is associated with poor outcomes. As clinico-radiological findings and pathological features are often non-specific, the diagnosis of intimal sarcoma is challenging. Recently, MDM2 amplification was reported to be a characteristic genetic event in this tumor. In the present study, we examined MDM2 status by immunohistochemistry, and by fluorescence and dual-color in situ hybridization (FISH and DISH) using intimal sarcoma (10 tumors), angiosarcoma (5), pulmonary sarcomatoid carcinoma (p-SC) (14) and chronic pulmonary thrombosis (CPT) (3) to investigate MDM2 amplification for the diagnosis of intimal sarcoma. MDM2 and CDK4 were immunopositive in all 10 intimal sarcoma tumors, and high-level amplification of MDM2 was detected in eight tumors by both FISH and DISH. The other two tumors had polysomy of chromosome 12 and overexpression of p53 protein. Although MDM2 aberrations were observed in three p-SCs (two with amplification and one with polysomy), angiosarcomas and CPTs lacked MDM2 amplification. Furthermore, there was high concordance between FISH and DISH. In conclusion, we found that MDM2 amplification strongly supports the diagnosis of intimal sarcoma, and MDM2 DISH was a concordant method and an acceptable alternative to FISH. As MDM2 amplification and p53 overexpression were mutually exclusive, disruption of the MDM2-p53 pathway may be an essential genetic event for this malignant tumor.

5-Lipoxygenase in monocytes emerges as a therapeutic target for intimal hyperplasia in a murine wire-injured femoral artery.

Given the importance of leukotrienes in vascular inflammation induced by local tissue injury, this study investigated the role for 5-lipoxygenase (5-LO) in monocytes in the development of intimal hyperplasia. As a mechanistic study, the importance of monocyte 5-LO in monocyte-macrophage differentiation with subsequent infiltration in neointima was evaluated. In a mouse model of wire-injured femoral artery, intimal hyperplasia started as early as 2wks after injury, and luminal area and blood flow were reduced due to increased neointima formation. Time-dependent increases in macrophage infiltration were observed in neointima and showed a positive relationship with neointima volume. In 5-LO-deficient (KO) mice or wild-type (WT) mice treated with an inhibitor of 5-LO activating protein (MK886, 1 and 10mg/kg), intimal hyperplasia and macrophage infiltration into neointima were reduced, but monocyte adhesion to injured luminal surface was not inhibited, which suggested 5-LO participates in monocyte-macrophage differentiation. In an in vitro study, monocyte-macrophage differentiation was found to be increased by high mobility group box 1 protein (HMGB1), but this effect was attenuated in cells isolated from 5-LO-KO mice. Furthermore, macrophage infiltration and intimal hyperplasia were more prominent in 5-LO-KO mice transplanted with monocytes from WT mice than in 5-LO-KO mice transplanted with monocytes from 5-LO-KO mice. Taken together, it was suggested that 5-LO in monocytes played a pivotal role in monocyte-macrophage differentiation and subsequent infiltration of macrophage in neointima, leading to vascular remodeling after vascular injury.

Cytoglobin regulates blood pressure and vascular tone through nitric oxide metabolism in the vascular wall.

The identity of the specific nitric oxide dioxygenase (NOD) that serves as the main in vivo regulator of O2-dependent NO degradation in smooth muscle remains elusive. Cytoglobin (Cygb) is a recently discovered globin expressed in fibroblasts and smooth muscle cells with unknown function. Cygb, coupled with a cellular reducing system, efficiently regulates the rate of NO consumption by metabolizing NO in an O2-dependent manner with decreased NO consumption in physiological hypoxia. Here we show that Cygb is a major regulator of NO degradation and cardiovascular tone. Knockout of Cygb greatly prolongs NO decay, increases vascular relaxation, and lowers blood pressure and systemic vascular resistance. We further demonstrate that downregulation of Cygb prevents angiotensin-mediated hypertension. Thus, Cygb has a critical role in the regulation of vascular tone and disease. We suggest that modulation of the expression and NOD activity of Cygb represents a strategy for the treatment of cardiovascular disease.

Valsartan attenuates intimal hyperplasia in balloon-injured rat aortic arteries through modulating the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas receptor axis.

The role of the Mas receptor in the activity of valsartan against intimal hyperplasia is unclear. Herein, we investigated the role of the angiotensin-converting enzyme 2 (ACE2)-angiotensin-(1-7)-Mas receptor axis on the activity of valsartan against intimal hyperplasiain balloon-injured rat aortic arteries. Wistar rats were randomized equally into the sham control group, injured group, and injured plus valsartan (20 mg/kg/d)-treated group. Valsartan significantly attenuated the vascular smooth muscle cell proliferation and intimal and medial thickening on days 14 and 28 after injury. The angiotensin-(1-7) levels as well as ACE2 and Mas receptor mRNA/protein expression were significantly decreased in the injured rats, compared to the uninjured rats; meanwhile, the angiotensin II level as well as the ACE and AT1 receptor mRNA/protein expression were increased (all P < 0.05 or < 0.01). Additionally, the p-ERK protein expression was increased (P < 0.01). Treatment with valsartan significantly increased the angiotensin-(1-7) levels as well as ACE2 and Mas receptor mRNA/protein expression but decreased the angiotensin II level, ACE and AT1 receptor mRNA/protein expression, as well as the p-ERK protein expression, compared to the injured group (all P < 0.05 or < 0.01). These results suggest that valsartan attenuates neointimal hyperplasiain balloon-injured rat aortic arteries through activation of the ACE2-angiotensin-(1-7)-Mas axis as well as inhibition of the ACE-angiotensin II-AT1 and p-ERK pathways.

Anti-inflammatory and anti-proliferative effects of CBS3830 in arterialized vein grafts in rats.

OBJECTIVE: To investigated whether CBS3830, a highly selectively inhibitor of p38MAPK, could ameliorate inflammation and intimal hyperplasia in arterialized vein grafts (AVGs). METHODS: Sixty male Sprague-Dawley rats underwent a reversed right jugular vein to common carotid artery interposition graft and were randomly treatment with vehicle (control) or single-dose (3 mg/kg, preoperative) or double-dose (3 mg/kg, preoperative and 4 d postoperative) CBS3830. Twenty rats underwent sham operation. The levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were determined by ELISA. Vein grafts were analyzed by intimal/medial morphometry, proliferating cell nuclear antigen (PCNA) expression, and p38MAPK phosphorylation. RESULTS: TNF-α, IL-1ß, and IL-6 gradually increased then slowly decreased in AVG rats. However, at 4 d and 7 d, TNF-α levels decreased by 37.5% and 29.5% (p = 0.003, 0.05, respectively) in the single-dose CBS3830 group, and by 37.6% and 32.5%, respectively (both p = 0.003) in the double-dose group compared with those of control. IL-1ß levels significantly reduced at 4 d and 14 d in both dosage groups. IL-6 levels significantly reduced at 7 d in both groups. Intima and medial thickening were significantly reduced in both dosage treated groups at 7, 14, and 28 d (all p = 0.000) compared to the controls. Further study showed CBS3830 inhibited p38MAPK phosphorylation and decreased PCNA expression. CONCLUSIONS: CBS3830 significantly decreases inflammation and intimal hyperplasia in AVGs.

p38 MAPK inhibitor, CBS3830 limits vascular remodelling in arterialised vein grafts.

OBJECTIVE: Following bypass surgery vein grafts undergo a remodelling process that can lead to restenosis and ultimately vein graft failure. Signalling through mitogen activated protein kinases (MAPKs) is a key mechanism involved in vein graft failure. Here, we investigated whether CBS3830 (c-a-i-r biosciences GmbH, Tübingen, Germany), a new highly selectively inhibitor of p38 MAPK, has a significant effect on inhibiting intimal, medial and adventitial hyperplasia. METHODS: Sixty specific pathogen free Sprague Dawley male rats were randomly divided into three groups. The control group with a reversed right jugular vein, which is common to carotid artery interposition graft, was compared with sham-operated, and CBS3830 treated animals. Intimal, medial and adventitia morphometric examinations and expression of proliferating cell nuclear antigen (PCNA) were analysed after one, two and four weeks for vein grafts. RESULTS: Intimal, medial and adventitia thickening in CBS3830 group were significantly lower than in the control group at each time point. Moreover, CBS3830 significantly reduced the phosphorylation of p38 MAPK and PCNA expression compared to the control. CONCLUSION: On the basis of the present work, intima, media and adventitia of saphenous vein grafts undergo vascular remodelling after surgery. The new, highly selective p38 MAPK inhibitor, CBS3830, ameliorates intimal, medial, and adventitial remodelling by varying degrees.

Hydrogen sulfide in the mouse ductus arteriosus: a naturally occurring relaxant with potential EDHF function.

We have previously reported that bradykinin relaxes the fetal ductus arteriosus via endothelium-derived hyperpolarizing factor (EDHF) when other naturally occurring relaxants (prostaglandin E2, nitric oxide, and carbon monoxide) are suppressed, but the identity of the agent could not be ascertained. Here, we have examined in the mouse whether hydrogen sulfide (H2S) is a relaxant of the ductus and, if so, whether it may also function as an EDHF. We found in the vessel transcripts for the H2S synthetic enzymes, cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS), and the presence of these enzymes was confirmed by immunofluorescence microscopy. CSE and CBS were distributed across the vessel wall with the former prevailing in the intimal layer. Both enzymes occurred within the endoplasmic reticulum of endothelial and muscle cells, whereas only CSE was located also in the plasma membrane. The isolated ductus contracted to inhibitors of CSE (d,l-propargylglycine, PPG) and CBS (amino-oxyacetic acid), and PPG contraction was attenuated by removal of the endothelium. EDHF-mediated bradykinin relaxation was curtailed by both PPG and amino-oxyacetic acid, whereas the relaxation to sodium nitroprusside was not affected by either treatment. The H2S donor sodium hydrogen sulfide (NaHS) was also a potent, concentration-dependent relaxant. We conclude that the ductus is endowed with a H2S system exerting a tonic relaxation. In addition, H2S, possibly via an overriding CSE source, qualifies as an EDHF. These findings introduce a novel vasoregulatory mechanism into the ductus, with implications for antenatal patency of the vessel and its transitional adjustments at birth.

Role for Gßγ G-proteins in protease regulation during remodeling of the murine femoral artery.

BACKGROUND: Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. G-protein coupled receptors are involved in smooth muscle cell proliferation but the role of Gßγ in arterial intimal hyperplasia has not been well defined. The aim of this study is to characterize the expression of Gßγ G-proteins in the developing intimal hyperplasia in a murine model and the impact of disruption of Gßγ signaling on intimal hyperplasia development. METHODS: The murine femoral wire injury model was employed. Specimens were perfusion-fixed and sections were stained with H&E and Movat's stains such that morphometry could be performed using an Image-Pro system. Additional specimens of femoral artery were also harvested and snap frozen for Western blotting for the Gßγ expression and for Western blotting and zymography to allow for the study of gelatinase and plasminogen activator expression and activation. Contralateral vessels were used as controls. Additional vessels were immersed in pluronic gel containing an adenovirus with the Gßγ inhibitor ßARK(CT). RESULTS: The injured femoral arteries developed intimal hyperplasia, while sham vessels did not produce such a response. Cell proliferation peaked at 3-5 d and cell migration at 7 d after injury. There was a marked time-dependent increase in Gßγ over the 28 d following injury. Inhibition of Gßγ with ßARK(CT) inhibited cell proliferation, cell migration and the development of intimal hyperplasia. Inhibition of Gßγ decreased peak uPA activity and expression without increasing early PAI-1 activity and expression. Inhibition of Gßγ reduced peak MMP-2 activity at d 1 but not at d 7 and also reduced peak MMP-9 activity at d 3. Protein expression for both MMP-2 and MMP-9 was also transiently decreased. There were no changes in TIMP-1 and TIMP-2 expression and activity. CONCLUSIONS: These data demonstrate a time-dependent increase in Gßγ G-protein expression following wire injury in the mouse. Inhibition of Gßγ alters cell proliferation and migration with associated changes in MMP-2, MMP-9, and uPA expression and activity.

Role of MT1-MMP in estrogen-mediated cellular processes of intimal hyperplasia.

BACKGROUND: Hormone replacement therapy increases intimal hyperplasia (IH) following vascular intervention. Matrix metalloproteinases (MMPs) play a role in IH development. We have shown estrogen up-regulates MT1-MMP expression, a transmembrane protein that activates MMP-2, and increases vascular smooth muscle cell (VSMC) collagen invasion via increased MMP-2 activity. Here we hypothesize inhibition of MT1-MMP will prevent hormonally-stimulated increased MMP-2 activation and the downstream cellular processes of IH pathogenesis. METHODS: VSMCs from a postmenopausal donor were transfected with MT1-MMP or negative control siRNAs, treated with estrogen (Est), analyzed by q-PCR, Western blot, zymography, migration, invasion, and proliferation assays. RESULTS: Est treatment of MT1-MMP silenced cells still resulted in increased MT1-MMP expression (C = 41% ± 4%; Est = 52% ± 2%; P < 0.05). Silencing of MT1-MMP decreased basal MMP-2 activity (nonsilenced = 100%; MT1-silenced = 87% ± 3%; P < 0.05) but had no effect on basal invasion or proliferation. Est treatment of MT1-MMP silenced cells still resulted in increased MMP-2 activity (C = 87% ± 3%; Est = 101% ± 4%; P < 0.05) and invasion (C = 89% ± 6%; Est = 109% ± 3%; P < 0.05) compared with MT1-MMP silenced control cells. However, silencing of MT1-MMP did inhibit Est- and serum-stimulated proliferation (C = 106% ± 18%; Est = 104% ± 16%; FBS = 121% ± 24%; P = NS). CONCLUSION: Silencing of MT1-MMP in aged VSMCs results in impaired but not complete inhibition of basal and Est-stimulated increases in MMP-2 activity. Other mechanisms appear to be playing a role in hormonally-regulated cellular processes of IH pathogenesis. Future studies will target other signaling cascades, with the goal of identifying mechanisms responsible for hormonally-modulated unbalanced MMPs. In vivo manipulation of the expression patterns of MT1-MMP will be examined for the prevention of IH in animal models of vascular disease.

Inhibition of Mitogen Activated Protein Kinase Activated Protein Kinase II with MMI-0100 reduces intimal hyperplasia ex vivo and in vivo.

Vein graft intimal hyperplasia remains the leading cause of graft failure, despite many pharmacological approaches that have failed to translate to human therapy. We investigated whether local suppression of inflammation and fibrosis with MMI-0100, a novel peptide inhibitor of Mitogen Activated Protein Kinase Activated Protein Kinase II (MK2), would be an alternative strategy to reduce cell proliferation and intimal hyperplasia. The cell permeant peptide MMI-0100 was synthesized using standard Fmoc chemistry. Pharmacological doses of MMI-0100 induced minimal human endothelial and smooth muscle cell proliferation (30% and 12% respectively). MMI-0100 suppressed IL-6 expression to control levels, without effect on IL-8 expression. MMI-0100 caused sodium nitroprusside induced smooth muscle cell relaxation and inhibited intimal thickening in human saphenous vein rings in a dose-dependent fashion. In a murine aortic bypass model, MMI-0100 reduced intimal thickness in vein grafts by 72%, and there were fewer F4/80-reactive cells in vein grafts treated with MMI-0100. MMI-0100 prevents vein graft intimal thickening ex vivo and in vivo. These results suggest that inhibition of MK2 with the cell-permeant peptide MMI-0100 may be a novel strategy to suppress fibrotic processes such as vein graft disease.

AMPKα2 deletion exacerbates neointima formation by upregulating Skp2 in vascular smooth muscle cells.

RATIONALE: Adenosine monophosphate-activated protein kinase (AMPK), a metabolic and redox sensor, is reported to suppress cell proliferation of nonmalignant and tumor cells. Whether AMPKα alters vascular neointima formation induced by vascular injury is unknown. OBJECTIVE: The aim of this study was to determine the roles of AMPKα in the development of vascular neointima hyperplasia and to elucidate the underlying mechanisms. METHODS AND RESULTS: Vascular smooth muscle cell (VSMC) proliferation and neointimal hyperplasia were evaluated in cultured VSMCs and wire-injured mouse carotid arteries from wild-type (WT, C57BL/6J), AMPKα2(-/-), and AMPKα1(-/-) mice. Mouse VSMCs derived from aortas of AMPKα2(-/-) mice exhibited increased proliferation compared with either WT or AMPKα1(-/-) VSMCs. Further, deletion of AMPKα2 but not AMPKα1 reduced the level of p27(Kip1), a cyclin-dependent kinase inhibitor, and increased the level of S-phase kinase-associated protein 2 (Skp2), a known E3 ubiquitin ligase for p27(Kip1), through activation of p52 nuclear factor kappa B (NF-κB)-2. Moreover, either pharmacological (ie, through compound C) or genetical (ie, through AMPKα2-specific siRNA) inhibition of AMPK decreased p27(Kip1) levels but increased the abundance of Skp2 in human VSMCs. Furthermore, gene silencing of Skp2 reversed the levels of p27(Kip1) and VSMCs proliferation. Finally, neointima formation after mechanical arterial injury was increased in AMPKα2(-/-) but not AMPKα1(-/-) mice. CONCLUSIONS: These findings indicate that deletion of AMPKα2 through p52-Skp2-mediated ubiquitination and degradation of p27(Kip1) accentuates neointimal hyperplasia in response to wire injury.

Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK via PKCα-mediated MKP-1 induction.

AIMS: Hyperplasia of vascular smooth muscle cells (VSMCs) after blood vessel injury is one of the major pathophysiological mechanisms associated with neointima. Tauroursodeoxycholate (TUDCA) is a cytoprotective agent in a variety of cells including hepatocytes as well as an inducer of apoptosis in cancer cells. In this study, we investigated whether TUDCA could prevent neointimal hyperplasia by suppressing the growth and migration of VSMCs. METHODS AND RESULTS: Transporters of TUDCA uptake in human VSMCs (hVSMCs) were analysed by RT-PCR and western blot. A knock-down experiment using specific si-RNA revealed that TUDCA was incorporated into hVSMCs via organic anion transporter 2 (OATP2). TUDCA reduced the viability of hVSMCs, which were mediated by inhibition of extracellular signal-regulated kinase (ERK) by induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) via protein kinase Cα (PKCα). The anti-proliferative effect of TUDCA was reversed by treatment with 7-hydroxystaurosporine, an inhibitor of PKC, and by the knock-down of MKP-1. In addition, TUDCA suppressed hVSMC migration, which was mediated by reduced matrix metalloproteinase-9 (MMP-9) expression by ERK inhibition, as well as reduced viability of hVSMCs. Rats with carotid artery balloon injury received oral administration of TUDCA; this reduced the increase in ERK and MMP-9 caused by balloon injury. TUDCA significantly decreased the ratio of intima to media by reducing proliferation and inducing apoptosis of the VSMCs. CONCLUSION: TUDCA inhibits neointimal hyperplasia by reducing proliferation and inducing apoptosis of smooth muscle cells by suppression of ERK via PKCα-mediated MKP-1 induction.

Carotid intima-media thickness and paraoxonase activity in patients with ankylosing spondylitis.

PURPOSE: The risk of atherosclerosis is increased in several rheumatological disorders, but any such risk remains unproven for ankylosing spondylitis. Since carotid intima-media thickness is an indicator of early atherosclerosis, and the paraoxonase (PON1) enzyme has antioxidant activity to prevent LDL oxidation, we aimed to identify: 1) the relationship between carotid intima-media thickness (CIMT) and serum paraoxonase (PON1) activity in ankylosing spondylitis (AS) patients; and 2) the possible differences in CIMT in AS patients versus age-matched, healthy controls. METHODS: Forty-five AS patients (36.8±9.8 years, 36 males, 9 females) and 30 controls (35.9±10.2 years, 23 males, 7 females) were recruited consecutively. Serum PON1 activity and CIMT were measured. The Bath Ankylosing Spondylitis Metrology Index (BASMI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Radiologic Index (BASRI) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) were used to identify relationships between these clinical indices and levels of CIMT and PON1. RESULTS: Mean CIMT was significantly increased in AS patients relative to controls (0.49±0.06 mm vs. 0.59±0.07 mm; p < 0.0001). Conversely, serum PON1 activity was decreased (199.1±60.3 U/L vs. 96.7±29 U/L; p < 0.0001). PON1 activity was negatively correlated with CIMT (r = -0.557, p = 0.0001). Disease duration was positively correlated with CIMT (r = 0.542, p = 0.0001) and negatively correlated with PON1 (r = -0.649, p = 0.0001). On multivariate analysis, disease duration and serum PON1 activity were found to be independent predictors of CIMT (R2 = 0.687, p = 0.0001). CONCLUSIONS: In conclusion, significantly increased CIMT and decreased PON1 activity suggest a relationship between atherosclerosis and AS: a relationship that is strongly correlated with disease duration.

Role of IN-1233 in the prevention of neointimal hyperplasia after stent placement in a rat artery model.

PURPOSE: To evaluate the efficacy of an activin receptor-like kinase (ALK) 5 inhibitor, IN-1233, for the prevention of neointimal hyperplasia after bare stent placement in a rat common iliac artery (CIA) model. MATERIALS AND METHODS: All experiments were approved by the committee of animal research. A self-expanding metallic bare stent (2 mm × 6 mm) was inserted into the left CIA of 26 Sprague-Dawley male rats (300-360 g) under fluoroscopic guidance. IN-1233 was injected via the intraperitoneal route daily in 13 rats for 8 weeks after stent placement (group A); the other 13 rats underwent stent placement only (group B). Angiography was performed immediately and 4 weeks and 8 weeks after stent placement. Rats were sacrificed at 8 weeks after stent placement, and histologic findings were obtained. The neointimal area (NA), percentage of neointimal hyperplasia (%NH), and neointimal-to-medial area ratio (N/M) were assessed and compared between the two groups. RESULTS: Stent placement was technically successful. In 25 rats, arteries with stent placement were angiographically patent, whereas 1 rat in group B had an occlusion. The NA (0.31 mm(2) ± 0.09 vs 0.56 mm(2) ± 0.17; P < .001), the %NH (26.16% ± 8.75 vs 44.71% ± 17.75; P < .001) and the N/M (1.93 ± 0.77 vs 4.77 ± 2.26; P < .001) were significantly decreased in group A compared with group B. CONCLUSIONS: IN-1233 was shown in this study to be effective for the prevention of neointimal hyperplasia after bare metallic stent placement in a rat CIA model.

Overexpression of angiotensin II type 2 receptor suppresses neointimal hyperplasia in a rat carotid arterial balloon injury model.

Angiotensin II (ANG II) type 2 receptor (AT2R) has been recognized to suppress the proliferation of vascular smooth muscle cells (VSMCs). The aim of the present study was to determine whether AT2R overexpression inhibits neointimal hyperplasia in a rat carotid arterial balloon injury model and to examine the underlying mechanisms of its activity. Balloon-injured rats receiving Ad-AT2R showed significant diminutions in neointimal area and intima/media ratio compared to non-treated rats or rats receiving adenovirus containing green fluorescent protein (Ad-GFP). In addition, extracellular regulated kinase 1/2 (ERK1/2) and basic transcription element-binding protein 2 (BTEB2) were significantly down-regulated in the arteries and VSMCs of Ad-AT2R-treated rats and compared to Ad-GFP-treated rats. However, Ad-AT2R transfection failed to affect the expression of ANG II type 1 receptor (AT1R) in carotid arteries and cultured VSMCs. The present study provides direct evidence that AT2R plays a beneficial role in balloon injury-induced neointimal hyperplasia, which is mainly attributed to the inhibition of VSMC proliferation and involves the down-regulation of the ERK1/2 and BTEB2 pathways, but is independent of the expression of AT1R.

Augmented expression and activity of extracellular matrix-degrading enzymes in regions of low endothelial shear stress colocalize with coronary atheromata with thin fibrous caps in pigs.

Background- The molecular mechanisms that determine the localized formation of thin-capped atheromata in the coronary arteries remain unknown. This study tested the hypothesis that low endothelial shear stress augments the expression of matrix-degrading proteases and thereby promotes the formation of thin-capped atheromata. Methods and Results- Intravascular ultrasound-based, geometrically correct 3-dimensional reconstruction of the coronary arteries of 12 swine was performed in vivo 23 weeks after initiation of diabetes mellitus and a hyperlipidemic diet. Local endothelial shear stress was calculated in plaque-free subsegments of interest (n=142) with computational fluid dynamics. At week 30, the coronary arteries (n=31) were harvested and the same subsegments were identified. The messenger RNA and protein expression and elastolytic activity of selected elastases and their endogenous inhibitors were assessed. Subsegments with low preceding endothelial shear stress at week 23 showed reduced endothelial coverage, enhanced lipid accumulation, and intense infiltration of activated inflammatory cells at week 30. These lesions showed increased expression of messenger RNAs encoding matrix metalloproteinase-2, -9, and -12, and cathepsins K and S relative to their endogenous inhibitors and increased elastolytic activity. Expression of these enzymes correlated positively with the severity of internal elastic lamina fragmentation. Thin-capped atheromata developed in regions with lower preceding endothelial shear stress and had reduced endothelial coverage, intense lipid and inflammatory cell accumulation, enhanced messenger RNA expression and elastolytic activity of MMPs and cathepsins, and severe internal elastic lamina fragmentation. Conclusions- Low endothelial shear stress induces endothelial discontinuity and accumulation of activated inflammatory cells, thereby augmenting the expression and activity of elastases in the intima and shifting the balance with their inhibitors toward matrix breakdown. Our results provide new insight into the mechanisms of regional formation of plaques with thin fibrous caps.

Microsomal prostaglandin e2 synthase-1 modulates the response to vascular injury.

BACKGROUND: Microsomal (m) prostaglandin (PG) E2 synthase (S)-1 catalyzes the formation of PGE2 from PGH2, a cyclooxygenase product that is derived from arachidonic acid. Previous studies in mice suggest that targeting mPGES-1 may be less likely to cause hypertension or thrombosis than cyclooxygenase-2-selective inhibition or deletion in vivo. Indeed, deletion of mPGES-1 retards atherogenesis and angiotensin II-induced aortic aneurysm formation. The role of mPGES-1 in the response to vascular injury is unknown. METHODS AND RESULTS: Mice were subjected to wire injury of the femoral artery. Both neointimal area and vascular stenosis were significantly reduced 4 weeks after injury in mPGES-1 knockout mice compared with wild-type controls (65.6 ± 5.7 versus 37.7 ± 5.1 × 10³ pixel area and 70.5 ± 13.4% versus 47.7 ± 17.4%, respectively; P < 0.01). Induction of tenascin-C, a proproliferative and promigratory extracellular matrix protein, after injury was attenuated in the knockouts. Consistent with in vivo rediversion of PG biosynthesis, mPGES-1-deleted vascular smooth muscle cells generated less PGE2 but more PGI2 and expressed reduced tenascin-C compared with wild-type cells. Both suppression of PGE2 and augmentation of PGI2 attenuate tenascin-C expression and vascular smooth muscle cell proliferation and migration in vitro. CONCLUSIONS: Deletion of mPGES-1 in mice attenuates neointimal hyperplasia after vascular injury, in part by regulating tenascin-C expression. This raises for consideration the therapeutic potential of mPGES-1 inhibitors as adjuvant therapy for percutaneous coronary intervention.

Increased 12/15-lipoxygenase enhances cell growth, fibronectin deposition, and neointimal formation in response to carotid injury.

OBJECTIVE: To determine whether increased 12/15-lipoxygenase (12/15LO) expression in vivo enhances neointimal formation in response to injury. METHODS AND RESULTS: 12/15LO expression in the vessel wall is increased in animal models of metabolic syndrome and diabetes mellitus. Increased expression of 12/15LO enhances cultured vascular smooth muscle cell (VSMC) proliferation, an effect mediated by the helix-loop-helix factor inhibitor of differentiation 3 (Id3). Carotid endothelial denudation was performed on apolipoprotein (Apo) E(-/-), ApoE(-/-)/12/15LO(-/-), C57BL/6, and 12/15LO-overexpressing transgenic mice. ApoE(-/-)/12/15LO(-/-) mice had attenuated and 12/15LO-overexpressing transgenic mice had enhanced neointimal formation compared with control mice. 12/15LO-overexpressing transgenic mice had greater postinjury carotid Id3 and Ki-67 expression, cell number, and fibronectin deposition compared with C57BL/6 mice. Loss of 12/15LO attenuated proliferation of cultured ApoE(-/-) VSMCs, whereas 12/15LO overexpression induced VSMC proliferation. Loss of Id3 enhanced immunoglobulin trascription factor (ITF)-2b binding to and activation of the p21(cip1) promoter and abrogated 12/15LO-induced VSMC proliferation. CONCLUSIONS: To our knowledge, these data are the first demonstration that increased expression of 12/15LO in the vessel wall enhances Id3-dependent cell proliferation, fibronectin deposition, and neointimal formation in response to injury. Results identify p21(cip1) as a potential target of the 12/15LO-Id3 pathway and suggest that modulation of this pathway may have therapeutic implications for targeting the increased risk of restenosis in patients with diabetes.

Association of aorta intima permeability with myosin light chain kinase expression in hypercholesterolemic rabbits.

The development of hypercholesterolemia is a multifactorial process in which elevated plasma cholesterol levels play a central role. This study analyzed the variability of the expression and activity of myosin light chain kinase (MLCK) and endothelial permeability in the artery wall of rabbits after feeding the animals with a normal or a high-cholesterol diet. Hypercholesterolemia was induced by a high-cholesterol diet for 4 weeks. Aortas were removed and analyzed for endothelial permeability and MLCK expression. Samples of the arterial media were analyzed for MLCK activity and expression. A selective MLCK inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7) were used in hypercholesterolemia rabbit (1 mg/kg body weight). The aortas of high-cholesterol diet rabbits showed an increase in MLCK expression and activity (nearly threefold compare with control) as well as endothelial permeability. ML7 inhibit MLC phosphorylation and MLCK activity (nearly twofold compare with control) and endothelial permeability stimulated by cholesterol. These results indicate for the first time that hypercholesterolemia may be associated with MLCK expression and activity through which endothelial permeability is increased.