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Objective: Proliferation of smooth muscle cells (SMC) in response to vascular injury is central to neointimal vascular remodeling. There is accumulating evidence that histone acetylation constitutes a major epigenetic modification for the transcriptional control of proliferative gene expression; however, the physiological role of histone acetylation for proliferative vascular disease remains elusive., Methods and Results: In the present study, we investigated the role of histone deacetylase (HDAC) inhibition in SMC proliferation and neointimal remodeling. We demonstrate that mitogens induce transcription of HDAC 1, 2, and 3 in SMC. Short interfering RNA-mediated knockdown of either HDAC 1, 2, or 3 and pharmacological inhibition of HDAC prevented mitogen-induced SMC proliferation. The mechanisms underlying this reduction of SMC proliferation by HDAC inhibition involve a growth arrest in the G(1) phase of the cell cycle that is due to an inhibition of retinoblastoma protein phosphorylation. HDAC inhibition resulted in a transcriptional and posttranscriptional regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip). Furthermore, HDAC inhibition repressed mitogen-induced cyclin D1 mRNA expression and cyclin D1 promoter activity. As a result of this differential cell cycle-regulatory gene expression by HDAC inhibition, the retinoblastoma protein retains a transcriptional repression of its downstream target genes required for S phase entry. Finally, we provide evidence that these observations are applicable in vivo by demonstrating that HDAC inhibition decreased neointima formation and expression of cyclin D1 in a murine model of vascular injury., Conclusions: These findings identify HDAC as a critical component of a transcriptional cascade regulating SMC proliferation and suggest that HDAC might play a pivotal role in the development of proliferative vascular diseases, including atherosclerosis and in-stent restenosis.

Background: Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation., Methods and Results: Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibril-associated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls. Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin., Conclusions: Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta.

Background: Cardiovascular disease is the leading cause of mortality in chronic kidney disease patients on maintenance dialysis. Given the importance of matrix metalloproteinase-2 (MMP-2) in matrix integrity, vascular cell function, and structural stability, we hypothesized that MMP-2 was elevated in the macrovasculature in dialyzed chronic kidney disease patients compared with chronic kidney disease patients not on dialysis and kidney donors., Methods and Results: Arteries from live kidney donors (A(donor); n=30) and recipients (nondialysis [A(nondialyzed)], n=17; dialysis [A(dialyzed)], n=23 [peritoneal dialysis, n=10; hemodialysis, n=13]) were harvested during the transplantation procedure. Compared with A(donor), MMP-2 upregulation was evident in both recipient groups. Protein expression of latent plus active MMP-2 in A(dialyzed) was 2-fold that in A(nondialyzed). MMP-2 activity increased with length of dialysis (r=0.573, P=0.004). In A(dialyzed), medial elastic fiber fragmentation was pronounced, and the ratio of external elastic lamina to media was negatively correlated with MMP-2 activity (r=-0.638, P=0.001). A(dialyzed) was 25% stiffer than A(nondialyzed); this increased stiffness correlated with MMP-2 activity (r=0.728, P<0.0001) and the severity of medial calcium deposition (r=0.748, P=0.001). The contractile function and endothelium-dependent relaxation were reduced by 35% to 55% in A(dialyzed) and were negatively associated with MMP-2 activity (r=-0.608, P=0.002; r=-0.520, P=0.019, respectively). Preincubation with MMP-2 inhibitor significantly improved contractility and relaxation in A(dialyzed)., Conclusions: We describe a strong correlation between MMP-2 activation and elastic fiber disorganization, stiffness, calcification, and vasomotor dysfunction in the arterial vasculature in dialyzed chronic kidney disease patients. These findings may contribute to an improved understanding of mechanisms important in vascular health in chronic kidney disease patients.

Background and Purpose: Higher plasma concentrations of the endogenous nitric oxides synthase inhibitor asymmetrical dimethylarginine (ADMA) are associated with increased risk of cardiovascular and cerebrovascular events and death, presumably by promoting endothelial dysfunction and subclinical atherosclerosis. We hypothesized that plasma ADMA concentrations are positively related to common carotid artery intimal-media thickness (CCA-IMT) and to internal carotid (ICA)/bulb IMT., Methods: We investigated the cross-sectional relations of plasma ADMA with CCA-IMT and ICA/bulb IMT in 2958 Framingham Heart Study participants (mean age, 58 years; 55% women)., Results: In unadjusted analyses, ADMA was positively related to both CCA-IMT (beta per SD increment, 0.012; P<0.001) and ICA/bulb IMT (beta per SD increment, 0.059; P<0.001). In multivariable analyses (adjusting for age, sex, systolic blood pressure, antihypertensive treatment, smoking status, diabetes, BMI, total-to-HDL cholesterol ratio, log C-reactive protein, and serum creatinine), plasma ADMA was not associated with CCA-IMT (P=0.991), but remained significantly and positively related to ICA/bulb IMT (beta per SD increment, 0.0246; P=0.002)., Conclusions: In our large community-based sample, we observed that higher plasma ADMA concentrations were associated with greater ICA/bulb IMT, but not with CCA-IMT. These data are consistent with the notion that ADMA promotes subclinical atherosclerosis in a site-specific manner, with a greater proatherogenic influence at known vulnerable sites in the arterial tree.

We have recently demonstrated that stem cell antigen 1-positive (Sca-1(+)) progenitors exist in the vascular adventitia of apolipoprotein E-deficient (apoE(-/-)) mice and contribute to smooth muscle cell (SMC) accumulation in vein graft atherosclerosis. Using a combined proteomic and metabolomic approach, we now characterize these local progenitors, which participate in the formation of native atherosclerotic lesions in chow-fed apoE(-/-) mice. Unlike Sca-1(+) progenitors from embryonic stem cells, the resident Sca-1(+) stem cell population from the vasculature acquired a mature aortic SMC phenotype after platelet-derived growth factor-BB stimulation. It shared proteomic and metabolomic characteristics of apoE(-/-) SMCs, which were clearly distinct from wild-type SMCs under normoxic and hypoxic conditions. Among the differentially expressed proteins were key enzymes in glucose metabolism, resulting in faster glucose consumption and a compensatory reduction in baseline interleukin-6 secretion. The latter was associated with a marked upregulation of insulin-like growth factor binding proteins (IGFBPs) 3 and 6. Notably, reconstitution of interleukin-6 to levels measured in the conditioned medium of wild-type SMCs attenuated the elevated IGFBP expression in apoE(-/-) SMCs and their vascular progenitors. This coregulation of apoE, interleukin-6, and IGFBPs was replicated in wild-type SMCs from hypercholesterolemic mice and confirmed by silencing apoE expression in SMCs from normocholesterolemic mice. In summary, we provide evidence that Sca-1(+) progenitors contribute to native atherosclerosis in apoE(-/-) mice, that apoE deficiency and hypercholesterolemia alter progenitor cell behavior, and that inflammatory cytokines such as interleukin-6 act as metabolic regulators in SMCs of hyperlipidemic mice.

Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O(2)) or normoxic (21% O(2)) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension.

Dimethylarginie dimethylaminohydrolase (DDAH) degrades asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) synthase inhibitor, and comprises 2 isoforms, DDAH1 and DDAH2. To investigate the in vivo role of DDAH2, we generated transgenic mice overexpressing DDAH2. The transgenic mice manifested reductions in plasma ADMA and elevations in cardiac NO levels but no changes in systemic blood pressure (SBP), compared with the wild-type mice. When infused into wild-type mice for 4 weeks, ADMA elevated SBP and caused marked medial thickening and perivascular fibrosis in coronary microvessels, which were accompanied by ACE protein upregulation and cardiac oxidative stress. The treatment with amlodipine reduced SBP but failed to ameliorate the ADMA-induced histological changes. In contrast, these changes were abolished in transgenic mice, with a reduction in plasma ADMA. In coronary artery endothelial cells, ADMA activated p38 MAP kinase and the ADMA-induced ACE upregulation was suppressed by p38 MAP kinase inhibition by SB203580. In wild-type mice, long-term treatment with angiotensin II increased plasma ADMA and cardiac oxidative stress and caused similar vascular injury. In transgenic mice, these changes were attenuated. The present study suggests that DDAH2/ADMA regulates cardiac NO levels but has modest effect on SBP in normal conditions. Under the circumstances where plasma ADMA are elevated, including angiotensin II-activated conditions, ADMA serves to contribute to the development of vascular injury and increased cardiac oxidative stress, and the overexpression of DDAH2 attenuates these abnormalities. Collectively, the DDAH2/ADMA pathway can be a novel therapeutic target for vasculopathy in the ADMA or angiotensin II-induced pathophysiological conditions.

Background: Progressive medial degeneration and atrophy is thought to be a cause of ascending thoracic aortic aneurysms in the elderly. Extensive apoptosis of vascular smooth muscle cells (VSMCs) has been demonstrated in the media of abdominal aortic aneurysms. We investigated whether medial atrophy from loss of VSMCs occurs in primary ascending thoracic aortic aneurysms., Methods and Results: Morphometric analysis of 28 nonaneurysmal ascending thoracic aortas and 29 ascending thoracic aortic aneurysms was performed by directly measuring the thickness of their vascular layers and by indirectly calculating the area of their vascular compartments. The cellular and matrix composition of the media was assessed at the structural, protein, and transcript levels. Despite thinning of the media secondary to vascular dilatation, there was an overall increase in the medial area of aneurysms. VSMC density was preserved, implying cellular hyperplasia as a result of the increased medial mass. There was decreased expression of matrix proteins, despite sustained synthesis of these molecules, which was associated with evidence of increased matrix degradation. The remodeling and expansion of the media was most evident in comparisons between nonaneurysmal aortas versus smaller aneurysms and did not evolve further in larger aneurysms., Conclusions: The mechanisms for luminal enlargement in thoracic and abdominal aortic aneurysms differ significantly with regard to the survival of VSMCs and atrophy of the media but share common pathophysiology involving degeneration of the matrix. Hyperplastic cellular remodeling of the media in ascending thoracic aortic aneurysms may be an initial adaptive response to minimize increased wall stress resulting from vascular dilatation.

We previously showed that a systemic inhibitor of gp91(phox) (nox2)-based NAD(P)H oxidase abolishes angiotensin II (Ang II)-induced vascular hypertrophy. In the present study, we tested whether perivascular transfection with Ad-gp91ds-eGFP (an adenoviral bicistronic construct targeting NAD(P)H oxidase in fibroblasts) or controls Ad-CMV-eGFP and Ad-scrmb-eGFP would affect medial hypertrophy in response to Ang II. In C57BL/6J mice, we applied Ad-gp91ds-eGFP or controls to the left carotid adventitia, and 2 days later we implanted minipumps delivering vehicle or Ang II (750 microg/kg per day) for 7 days. None of the viral treatments affected Ang II-induced systolic blood pressure elevation. Immunohistochemical staining showed marker eGFP in adventitial fibroblasts and some macrophages, indicating expression of the gp91ds inhibitor. As expected, Ang II induced medial hypertrophy (medial cross-sectional area, 32.96+/-2.04 versus 20.57+/-1.00x10(3) microm2, Ang II versus control; P<0.001) that was significantly inhibited by Ad-gp91ds-eGFP (26.23+/-0.90x10(3) microm2; P<0.01) but not control viruses. Application of viruses alone did not change medial size under control conditions. Immunohistochemical staining and semiquantitative analysis showed a 70% increase in reactive oxygen species levels measured by the lipid peroxidation byproduct 4-hydroxynonenal (4-HNE) throughout the carotid wall in the Ang II group versus vehicle. After treatment with Ad-gp91ds-eGFP, 4-HNE generation was normalized. Thus NAD(P)H oxidases in adventitial fibroblasts and macrophages appear to modulate Ang II-induced medial hypertrophy.

Background: 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., Methods and Results: 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., Conclusions: 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.

Background: The tissue inhibitor of metalloproteinases-1 (TIMP-1) is expressed in atherosclerotic lesions, where it may play a critical role in regulating the activity of matrix metalloproteinases (MMPs). Several MMPs are overexpressed in the atherosclerotic plaque, and they are believed to contribute to the expansion and rupture of the lesion., Methods and Results: The Timp-1-knockout mouse model (Timp-1-/-) was crossed into the apolipoprotein E-knockout (apoE0) background. A study population of male apoE0 mice, half of them deficient in TIMP-1, was fed an atherogenic diet. After 10 weeks of the diet, the mean lesion sizes of the two groups of animals were not significantly different, and the average content of fibrillar collagen and macrophages in the lesions was similar. There was no sign of plaque hemorrhage, even after 22 weeks of high-fat diet, indicating that deficiency in TIMP-1 does not predispose to luminal rupture. However the atherosclerotic lesions of the Timp-1-/0 mice developed more aortic medial ruptures, in which all elastic lamellae of the media were degraded and infiltrated with macrophages, forming pseudo-microaneurysms. After 10 weeks of high-fat diet, the Timp-1-/0/apoE0 mice averaged 1.9+/-1.2 medial ruptures in the proximal aorta, compared with 0.5+/-0.7 for the apoE0 controls (P<0.003). At the site of degradation, in situ zymography revealed that the gelatinolytic activity, mainly associated with macrophages, could be abolished by the addition of MMP inhibitors., Conclusions: These data strongly suggest that TIMP-1 plays a key role in preventing medial degradation associated with atherosclerosis through its ability to inhibit the MMPs that are involved in the disruption of the media.

Objective: Some of the statin-induced reduction in cardiac events in patients with atherosclerosis may be derived from mechanisms independent of lipid lowering. This study tested in nonhuman primates whether statins can influence inflammation (indicated by vascular cell adhesion molecule-1, interleukin-1beta, tissue factor, and macrophages) and features of plaque stability (indicated by collagen and smooth muscle cells) independent of their effect on plasma cholesterol level., Methods and Results: Adult male cynomolgus monkeys (n=12 per group) consumed an atherogenic diet for 12 months while receiving (1) no treatment (control), (2) pravastatin (Prava, 40 mg/kg per day), or (3) simvastatin (Simva, 20 mg/kg per day). Dietary cholesterol was adjusted to equalize plasma cholesterol levels among groups. Although the intima/media ratio in the abdominal aorta did not differ among groups, drug treatment reduced inflammation and features of plaque vulnerability. Macrophage content in the lesions of statin-treated animals was lowered (2.4-fold with Prava and 1.3-fold with Simva; both P<0.001 versus control). Furthermore, lesions had approximately 2-fold less vascular cell adhesion molecule-1, interleukin-1beta, and tissue factor expression in statin-treated versus control animals (P<0.005). Lesional smooth muscle cell and collagen content was 2.1-fold greater in the Prava-treated group (P<0.001) and 1.5-fold greater in the Simva-treated group (P<0.005) than in the control group., Conclusions: In primates, these results provide further support for the beneficial effect of statins on plaque inflammation and stability in addition to cholesterol lowering.

Skeletal muscle and myocardium express microNOS I, an elongated splice variant of neuronal-type nitric oxide (NO) synthase (NOS I), and NOS III, endothelial-type NO synthase, respectively. This study was designed to elucidate whether vascular smooth muscle also contains a constitutively expressed NO synthase isoform. In the rat, microNOS I contains an insert of 102 nucleotides after nucleotide 2865 of the cDNA, yielding a protein of 164 kd. Reverse transcription-polymerase chain reaction with primers flanking this insert and with insert-specific primers indicated that endothelium-denuded rat aorta expresses both brain-type NOS I and microNOS I. RNase protection analyses with an antisense RNA probe overlapping the microNOS I insert detected significant amounts of NOS I mRNA and lesser amounts of microNOS I mRNA in endothelium-denuded aorta. Western blots using a specific polyclonal antibody recognizing NOS I and microNOS I showed a major band of the 160-kd NOS I and a lesser band of a slightly larger protein in endothelium-denuded aorta. Immunohistochemistry demonstrated low levels of NOS I/microNOS I immunoreactivity in the medial layer of rat aorta, whereas the endothelium expressed only NOS III immunoreactivity. When the adventitia also was removed, NOS I and microNOS I mRNA decreased markedly but remained detectable in the medial layer. In functional experiments with endothelium-denuded rat aortic rings (that contained no NOS III), contractions induced by KCl were markedly increased in the presence of the NOS inhibitor N(G)-nitro-L-arginine. These data demonstrate that 2 subforms of NOS I are expressed in nonendothelial components of rat aorta: NOS I and lesser amounts of microNOS I. Under certain conditions, this NOS I/microNOS I expression could serve as a backup system to the functionally predominant NOS III.

Background: 15-Lipoxygenase (15-LO) may be involved in atherogenesis and in oxidative modification of LDL. In this study, we investigated 15-LO expression in developing atherosclerotic lesions and verified the exact type of the atherosclerosis-associated LO at the nucleotide level., Methods and Results: Quantitative reverse transcription-polymerase chain reaction, in situ hybridization, and immunocytochemistry were used in two models of experimental atherosclerosis. New Zealand White rabbits were given a 1% cholesterol diet for 0 (control group), 3, 6, or 14 weeks. 15-LO mRNA was undetectable in the aortic intima-medias of the control group, whereas it was clearly induced as early as after 3 weeks. 15-LO expression increased further in the 6- and 14-week groups. According to in situ hybridization and immunocytochemical studies, 15-LO was localized to macrophagerich areas. In Watanabe heritable hyperlipidemic rabbits, 15-LO mRNA was undetectable in normal aortic intima-medias. 15-LO mRNA was markedly induced in fatty streaks but less so in more advanced lesions. Identification of the induced LO as reticulocyte-type 15-LO was done by cloning and sequencing. We also observed a distinct basal expression of copper-zinc and extracellular superoxide dismutases in normal aortic intima-medias, but no clear induction of these mRNAs was detected in atherosclerotic aortas., Conclusions: The results show that, in contrast to copper-zinc and extracellular superoxide dismutases, the expression of reticulocyte-type 15-LO is markedly induced in rabbit fatty streaks. This may lead to an increase in the oxidative potential during the early phase of atherogenesis and contribute to the development of atherosclerotic lesions.