Number Of Smooth Muscle Cells Research Articles

Medroxyprogesterone Abrogates the Inhibitory Effects of Estradiol on Vascular Smooth Muscle Cells by Preventing Estradiol Metabolism

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA.

Aortic Aneurysms

In the United States, aortic aneurysms are the 13th leading cause of deathsee 1, 2 About 15,000 individuals die every year due to the rupture of aortic aneurysms. Based on autopsy studies it has been estimated that 1-2% of the population harbor aneurysms in their aorta with up to 10% prevalence in older age groups.1, 2 Most aortic aneurysms go undetected until rupture and the mortality from ruptured aneurysms is as high as 90%.1, 2 Along the length of the aorta, significant heterogeneity occurs in the distribution of aneurysm disease. The prevalence of abdominal aortic aneurysms (AAAs) located in the infrarenal section of the aorta is at least three times higher than that of thoracic aortic aneurysms and dissections (TAAD).1, 2 In TAAs, about 50% involve the ascending aorta, 10% the arch and 40% the descending thoracic aorta.1, 2 Only about 25% of patients with TAAD have a concomitant AAA, and multisegmental disease is found in only about 10% of cases.1, 2 There are also other differences between TAAD and AAA: 1) age-at-onset for TAAD (65 years) is approximately 10 years earlier than for AAA (75 years); and 2) AAAs are predominantly a disease of Caucacian males with 6:1 male:female ratio, whereas TAADs occur only slightly more frequently in males (1.7:1). Additional differences can be found in the pathobiology of these aneurysms. AAAs are characterized by signs of local chronic inflammation of the aortic wall, decrease in the number of smooth muscle cells in the aortic media layer and fragmentation of the extracellular matrix of the aorta at the site of the aneurysm see 3. Increased local expression of proinflammatory cytokines and matrix metalloproteinases (MMPs) have also been demonstrated.3 Furthermore, AAAs can be induced in a surgical experimental model in which elastases are infused into rodent aorta.4 TAADs are characterized by medial necrosis also known as “Erdheim’s cystic medial necrosis” and more recently referred to as “medial degeneration”, mucoid infiltration, and cyst formation with elastin degradation and vascular smooth muscle cell apoptosis.1 Both TAAD and AAA are silent diseases often without symptoms.2 They can, however, be readily identified through imaging techniques. TAADs can be detected by echocardiography or CT, and family members of TAAD-patients will benefit from imaging by identifying their TAADs before catastrophic consequences. Presently, there are no recommendations about large scale screening of populations for TAAD. On the other hand, for AAA ultrasonography screening studies have demonstrated cost-effectiveness of population-based ultrasonography screening programs and a decrease in the number of aneurysm-related deaths.see,5 Several recommendations have been made including a recent consensus statement, in which Kent et al.6 recommended ultrasonography screening for AAA for all individuals over 60 years of age and for those over 50 years of age with family history for AAA and the recommendation by the US Preventive Service Task Force7 to screen for AAA in men aged 65 to 75 years who have ever smoked.

Fosinopril Prevents the Pulmonary Arterial Remodeling in Sinoaortic-denervated Rats by Regulating Phosphodiesterase

To study the effects of fosinopril on sinoaortic denervation (SAD)-induced pulmonary vascular remodeling and on phosphodiesterases (PDE) 1 in rats. SAD was performed in male Sprague-Dawley rats at the age of 10 weeks. The experiment included sham-operated (Sham), SAD, and fosinopril-treated SAD groups. Fosinopril (15 mg/kg/d) was given in rat chow. After 16 weeks of treatment, the pulmonary arteries were taken for investigations, including pharmacological study, measurement of cGMP, light microscopy, immunohistochemistry, Western blotting, and quantitative real-time RT-PCR. Compared with Sham rats, blood pressure variability (BPV) was significantly increased in the SAD group. However, the mean pulmonary artery pressure (mPAP) was not significant change among 3 groups. After SAD, maximal contraction of pulmonary artery rings to phenylephrine was markedly decreased; the most prominent morphological change in the lung included thickening vascular walls, increasing number of smooth muscle cells, and greater wall-to-lumen ratio; the tissue concentrations of cGMP was reduced significantly; PDE1A or PDE1C expression was upregulated significantly, and endothelial nitric oxide synthase (eNOS) expression was downregulated significantly. Fosinopril treatment prevented these changes induced by SAD. Pulmonary artery remodeling (structural and functional abnormalities) was induced by SAD. Fosinopril, an angiotensin-converting enzyme inhibitor, mainly via potentiating eNOS pathway and inhibiting AngII formation, effectively prevented increased blood pressure variability and vascular remodeling of the pulmonary artery after SAD by regulating the activity levels or expression of eNOS, cGMP, and PDE1s.

High-Dose Granulocyte-Colony Stimulating Factor Promotes Neointimal Hyperplasia in the Early Phase and Inhibits Neointimal Hyperplasia in the Late Phase After Vascular Injury

Granulocyte-colony stimulating factor (G-CSF) affects injured arteries through early endothelialization. Some reports, however, have cautioned that the restenosis rate may increase after G-CSF injection. In the present study, high-dose G-CSF was administered to mice with vascular injury to clarify its effect. Mice were received daily subcutaneous injections of saline or a high dose (300 microg/kg) of G-CSF for 5 days after vascular injury. In the FACS analysis, CD34-/Sca-1-positive progenitor cells were more abundant in the G-CSF group (p<0.05). Neointimal hyperplasia was more evident in the G-CSF group at 1 week (p<0.05), whereas at 4 weeks it was more evident in the control group (p<0.01). TUNEL-positive cells in the arterial wall were more numerous in the G-CSF group at day 1 (p<0.01). CD34-positive cells were observed in the G-CSF group at 1 week. Re-endothelialization appeared earlier in the G-CSF group (at 4 weeks; p<0.01). An increased number of 1A4-positive smooth muscle cells were found in bone marrow cell culture treated with G-CSF. High-dose G-CSF induced neointimal proliferation through excessive inflammation and bone marrow cell mobilization in the early phase. In the late phase, however, it induced early re-endothelialization and thereby inhibited neointimal hyperplasia.

Adenovirus-Mediated Heme Oxygenase Transfer Inhibits Graft Arteriosclerosis in Rat Aortic Transplants

Purpose Heme oxygenase-1 (HO-1) has been implicated in graft protection because its induction is associated with long-surviving allografts. The aim of this study was to analyze the efficacy of adenovirus-mediated HO-1 overexpression to inhibit graft arteriosclerosis and estimate whether the protective role correlated with nuclear factor κB (NF = κB) expression and grafts apoptosis. Methods Aortic transplantation was performed using inbred Brown-Norway (BN) rats (RT 1n male) as donors and Lewis rats (RT 1l male) as recipients. The experiments were divided into four groups: group A, Lewis–Lewis ( n = 6), no treatment; group B, BN–Lewis ( n = 6), no treatment; group C, BN–Lewis ( n = 6), donor aorta perfused with buffer containing 4 × 10 10 pfu Ad-Null (the noncoding adenoviral vector); and group D, BN–Lewis ( n = 6), donor aorta perfused with buffer containing 4 × 10 10 pfu Ad-HO-1 (adenoviral vector coding for HO-1). All grafts were harvested at 60 days after transplantation. Reverse transcriptase polymerase chain reaction was performed to detect the expression of HO-1 gene. Hematoxylin and eosin staining was used to detect the morphology and cytology of the transplanted vessels. Intimal thickening was detected by Masson staining. Immunohistochemistry was used to detect the localization and expression of HO-1 and NF-κB and TUNEL assays to detect the apoptosis of the grafts. Results Gene transfer of HO-1 grafts using an adenoviral vector resulted in the expression of HO-1 protein in endothelium and adventitia. We observed that the intimal thickness in Ad-HO-1–treated aortas (11.11 ± 0.92 μm) was significantly thinner compared with untreated (85.20 ± 6.90 μm) or Ad-null treated (87.20 ± 6.20 μm) aortas ( P < .01). Immunohistology showed that treatment with Ad-HO-1 resulted in a significant reduction in leukocyte infiltration and a decreased number of vascular smooth muscle cells in the intima, compared with Ad-null–treated aortas. The levels of NF-κB and the number of apoptotic cells in Ad-HO-1–treated aortas showed significantly lower compared with Ad-null–treated arotas ( P < .05). Conclusion HO-1 prevented the development of graft arteriosclerosis in the rat aortic transplant model. The protective role of HO-1 in chronic rejection lesions seemed to correlate with downregulation of the expression of NF-κB and inhibition of apoptosis in the grafts.

Neointimal Hyperplasia in Early Arteriovenous Fistula Failure

Hemodialysis vascular access dysfunction currently is a huge clinical problem. Although arteriovenous fistulas (AVFs) are the preferred form of permanent dialysis access, they continue to have significant problems with early AVF failure. Although inadequate dilatation of the venous segment was believed to have a role in early AVF failure, the exact pathogenesis of early AVF failure is unknown despite the magnitude of the clinical problem. Case series. Hemodialysis patients. Stenotic venous segments from 4 patients with early AVF failure were subjected to a detailed histological, morphometric, and immunohistochemical analysis. All 4 patients had significant luminal stenosis, primarily as a result of eccentric neointimal hyperplasia. This was confirmed through morphometric analysis, which documented intima-media area and thickness ratios that were greater than unity. Cellular phenotyping studies showed that the majority of cells within the region of neointimal hyperplasia were myofibroblasts, with smaller numbers of contractile smooth muscle cells. We described only a limited number of specimens. We show for the first time that aggressive neointimal hyperplasia is present in venous segment specimens from patients with early AVF failure. Future therapies to address this problem will need to target this pathogenetic pathway.

Abstract 1175: Critical Role of Bone Marrow ASC (Apoptosis-associated Speck-like Protein Containing a CARD) in Neointimal Formation After Vascular Injury in Mice

Background: ASC is an adaptor protein that forms inflammasome complex and regulates caspase-1-dependent interleukin (IL)-1β and IL-18 generation; however, the role of ASC in the pathophysiology of cardiovascular diseases remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC −/− ) mice. Methods and Results: Wire-mediated vascular injury was produced in the femoral artery of ASC −/− and wild-type (WT: C57BL/6) mice. Immunohistochemical analysis revealed that ASC was markedly expressed in the infiltrated macrophages in the neointima. Histological analysis revealed that neointimal formation was significantly reduced in ASC −/− mice as compared to that in the WT mice (I/M ratio, p = 0.007). IL-1β and IL-18 were clearly expressed in the neointimal lesion of the WT mice but showed decreased expression in the lesion of ASC −/− mice. However, TUNEL staining showed no significant difference in apoptotic cells of the media between these mice. Further, no significant differences were observed in early reendothelialization (CD31) and the number of macrophages (F4/80) and smooth muscle cells (SMCs: αSMA) per unit of the neointima. Flow cytometry analysis also showed no difference in the number of peripheral CD34 + /Flk-1 + cells (EPCs) after injury. To determine the contribution of bone marrow cells, we developed 3 types of bone marrow transplantation (BMT Wild+Wild , BMT Wild+ASC−/− , and BMT ASC−/− → Wild ) mice. The neointimal formation in BMT ASC−/−+Wild mice mice was remarkably reduced as compared to that in BMT Wild+Wild (I/M ratio, p = 0.0003) and BMT Wild+ASC−/− mice (p = 0.015), indicating that bone marrow ASC is critical for neointimal formation. In vitro experiments showed that the lipopolysaccharide-induced production of inflammatory cytokines such as IL-1β, IL-6, MCP-1, and IFN-γ, but not TNF-α, in ASC −/− bone marrow cells was significantly decreased. Further, serum-stimulated proliferation activity and MAP kinase (ERK1/2 and p38) activation of ASC −/− SMCs were not impaired. Conclusions: These findings suggest that bone marrow-derived ASC is critical for the neointimal formation after vascular injury and identify ASC as a novel therapeutic target for cardiovascular diseases.

Abstract 724: Synectin is Required for Normal PDGF Secretion and Smooth Muscle Cell Basic Science Recruitment

Background: Synectin, a ubiquitously expressed scaffold and PDZ protein, has been shown to be a key regulator in the formation of arterial vasculature. Primary arterial endothelial cells isolated from synectin−/− mice exhibited defects in PDGF signaling that were not present in arterial synectin+/+ or either venous synectin+/+ or synectin−/− endothelial cells. Methods: To address the role of synectin in mural cell recruitment, retinas from synectin−/− (KO) and synectin+/+ (WT) mice were stained for isolectin and alpha-smooth muscle actin. To determine the mechanisms responsible for defective smooth muscle cells recruitment, primary arterial and venous endothelial (EC) and smooth muscle (SMC) cells were isolated from synectin+/+ and synectin−/− mice. Results: Examination of the retinal vasculature in synectin−/− mice demonstrated poor smooth muscle cell coverage of the forming arterial tree with a number of SMC cells sitting some distance away from the vessels, a defect reminiscent of retinal abnormalities observed in PDGF-B−/− mice. Western blot analysis of lysates from primary KO SMC demonstrated levels of PDGFR-β analogous to WT, although expression of PDGF-B was markedly reduced in lysates from arterial, but not venous, primary KO endothelial cells. Transduction of KO EC with an adenoviral synectin construct was sufficient to restore PDGF-B protein levels. Further, qPCR was used to determine whether defects in PDGF-B protein production were at the transcription or translation level. Interestingly, there was only a minor decrease in the amount of PDGF-B mRNA in KO compared to WT EC, suggesting that synectin deficiency has selectively affected post-transcriptional regulation of PDGF-B expression. Migration of WT and KO SMC in response to PDGF-B or conditioned media from WT and KO endothelial cells was also assessed to determine the cell type specificity of PDGF defects in KO mice. Conclusions: Synectin is required for post-transcriptional regulation of PDGF-B in endothelial cells. Deficits in PDGF-B secretion by synectin−/− endothelial cells result in inadequate smooth muscle recruitment and coverage of arterial vessels.

Avaliação quantitativa das fibras elásticas na doença pulmonar obstrutiva crônica

To quantify elastic fibers (EFs) and smooth muscle (SM) cells, as well as CD4+ and CD8+ T lymphocytes, in stable chronic obstructive pulmonary disease (COPD). Surgical specimens were obtained from 15 COPD patients, 18 smokers without airflow limitation, and 14 nonsmokers. Histological and immunohistochemical methods were employed in order to quantify EFs, SM cells, CD4+ T cells, and CD8+ T cells. There was no significant difference in EF numbers among the three groups (p > 0.05). The number of EFs per unit area of lung tissue (mm(2)) and the percentage of EFs in the lung tissue were similar among the three groups. The numbers of SM cells were found to be higher in the COPD patients than in the smokers (p = 0.003) or in the nonsmokers (p = 0.009). There was a tendency toward an increase in CD8+ T-cell counts in the COPD patients. In specimens collected from the COPD patients, CD4+ T-cell counts were lower than in those collected from the smokers (p = 0.015) or from the nonsmokers (p = 0.003). There was a weak correlation between CD4+ T-cell count and the ratio of forced expiratory volume in one second to forced vital capacity (r(2) = 0.003). The EF counts were similar among the three groups. Hypertrophy/hyperplasia of airway wall SM cells was found in the COPD patients and in the smokers, indicating that airway remodeling occurs in smokers. The CD4/CD8 ratio was lower in the COPD patients.

Low-density lipoproteins impair migration of human coronary vascular smooth muscle cells and induce changes in the proteomic profile of myosin light chain

High-risk atheromatous plaques contain significant extra- and intracellular lipid deposits and very low smooth muscle cell numbers in the intima. However, the mechanisms inducing vessel wall remodelling and high-risk plaque composition are unknown. Low-density lipoproteins (LDLs) infiltrate the vessel wall and become retained and aggregated (agLDL) in the intima by binding to extracellular matrix proteoglycans. The cellular responses triggered by agLDL are not fully understood. This study was designed to investigate the effects of agLDL on vascular remodelling and repair, specifically studying human coronary vascular smooth muscle cell (VSMC) functions. Using a wound repair VSMC model system, we have shown that agLDL significantly impair cell migration. Proteomic analysis revealed a differential phenotypic pattern of myosin light chain and lower levels of phosphorylated myosin regulatory light chain (P-MRLC) in agLDL-exposed VSMC. LDL also induced changes in the subcellular localization of P-MRLC, with dephosphorylation strongly evident on the front edge of agLDL-treated migrating cells. PMA, a strong inducer of myosin light chain (MLC) phosphorylation, significantly reduced the effects of agLDL in VSMC migration. Inhibition of MLC kinase with ML9 did not affect MRLC dephosphorylation already induced by agLDL. Our results indicate that LDLs impair human VSMC migration and wound repair after injury. agLDL, and to a lesser extent nLDL, induce dephosphorylation of MRLC and striking changes in the subcellular localization of P-MRLC, a cytoskeleton protein involved in VSMC migration kinetics.

Progesterone Receptor Regulates Bcl-2 Gene Expression through Direct Binding to Its Promoter Region in Uterine Leiomyoma Cells

Uterine leiomyomas are smooth muscle cell tumors that cause irregular uterine bleeding and pregnancy loss in many reproductive-age women. Progesterone stimulates their growth, whereas treatment with progesterone receptor (PR) antagonists or selective progesterone receptor modulators shrinks these tumors. Molecular mechanisms underlying these observations are unknown. Bcl-2 is a key protein that inhibits apoptosis. It was proposed that growth enhancement of leiomyoma cells by progesterone was mediated via bcl-2 induction. Here we test the hypothesis that PR regulates the bcl-2 gene by directly binding to its promoter. The pure progesterone agonist R5020 increased the total number of viable primary human leiomyoma smooth muscle (LSM) cells in culture. Progesterone or R5020 (10(-6) m) significantly increased bcl-2 mRNA levels after 2 and 4 h by 9.2- and 3.4-fold, respectively, in LSM cells. Transient transfection with deletion mutants of bcl-2 promoter showed that the -1281/-258-bp region conferred responsiveness to progesterone induction in the presence of PR-A. We identified a palindromic progesterone response element (PRE) at -553/-539 bp. EMSA showed that PR in nuclear extracts from LSM cells bound specifically to this PRE. Chromatin immunoprecipitation-PCR confirmed in situ recruitment of PR to the -629/-388-bp region bearing the PRE. In vivo, bcl-2 mRNA levels correlated significantly with total PR mRNA levels in leiomyoma tissues. Taken together, progesterone via PR interacts with the bcl-2 promoter to induce its expression in leiomyoma tissue. This may explain, in part, the progesterone-dependent enhancement of growth in uterine leiomyoma.

The Effect of Irreversible Electroporation on Blood Vessels

We present a pilot study on the long term effects of irreversible electroporation (IRE) on a large blood vessel. The study was motivated by the anticipated use of IRE for treatment of cancer tumors abutting large blood vessels. A sequence of 10 direct current IRE pulses of 3800 V/cm, 100 micros each, at a frequency of 10 pulses per second, were applied directly to the carotid artery in six rats. Measuring tissue conductivity during the procedure showed, as predicted, an increase in conductivity during the application of the pulse, which suggests that this measurement can be used to control the application of IRE. All the animals survived the procedure and showed no side effects. Histology performed 28 days after the procedure showed that the connective matrix of the blood vessels remained intact and the number of vascular smooth muscle cells (VSMC) in the arterial wall decreased with no evidence of aneurysm, thrombus formation or necrosis. Average VSMC density was significantly lower following IRE ablation compared with control (24 +/- 11 vs. 139 +/- 14, P<0.001), with no apparent damage to extra cellular matrix components and structure. In addition to the relevance of this study to treatment of cancer near large blood vessels these findings tentatively suggest that IRE has possible applications to treatment of pathological processes in which it is desired to reduce the proliferation of VSMC population, such as restenosis and for attenuating atherosclerotic processes in clinical important locations such as coronary, carotid and renal arteries.

Differential regulation of Akt, caspases and MAP kinases underlies smooth muscle cell apoptosis during aortic remodelling in SHR treated with amlodipine

The regression of aortic hypertrophy is initiated by a transient wave of smooth muscle cell (SMC) apoptosis in spontaneously hypertensive rats (SHR) treated with antihypertensive drugs, although the molecular pathways remain unclear. Enzymes involved in apoptosis regulation were examined daily during onset aortic remodelling in SHR treated with amlodipine (20 mg kg(-1) day(-1)). Significant reduction of aortic SMC number occurred by day 3 of amlodipine, reaching -13% at 28 days, followed by a significant regression of medial hypertrophy by day 5, reaching -13% at 28 days. ISOL-positive (apoptotic) SMC nuclei increased by 4.6-fold between days 2 and 4, in temporal correlation with the activation of caspase-8 (2.7-fold) at day 2 only, caspase-3 at days 3 and 4 (1.7-fold) and caspase-9 at day 3 only (3.1-fold). Akt phosphorylation, a pro-survival pathway, was reduced prior to apoptosis at day 1 (-52%) and until day 3. During the first 6 days of amlodipine treatment, significant reduction in phosphorylation of mitogen-activated protein (MAP) kinases was transient for p38 (-46% at day 3 only) but continuous for ERK1/2 after 3 days (-40%), and for JNK after 4 days (>-50%). Amlodipine inhibition of Akt occurred prior to and during SMC apoptosis induction, a process mediated by the early activation of caspase-8 followed by caspase-9 and -3 and associated with MAP kinase inhibition. These findings provide insights about the molecular pathways underlying SMC apoptosis leading to vascular remodelling during amlodipine treatment of hypertension.

Pathogenic Sequence for Dissecting Aneurysm Formation in a Hypomorphic Polycystic Kidney Disease 1 Mouse Model

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a multi-system disorder characterized by progressive cyst formation in the kidneys. Serious complications of ADPKD are intracranial and aortic aneurysms. The condition is mainly caused by mutations in the PKD1 or PKD2 gene. We have carefully analyzed vascular remodeling in hypomorphic Pkd1(nl/nL) mouse model with dissecting aneurysms in the aorta. Quantitative real-time polymerase chain reaction revealed that in the aorta the expression of normal Pkd1 is reduced to approximately 26%. Using (immuno)histochemistry we have characterized the pathogenetic sequence for dissecting aneurysm formation. The aorta shows regions with accumulation of matrix components between the elastin lamellae. This is followed by increased numbers of smooth muscle cells and locally weakening of the media. In the intima, accumulation of matrix components and detachment of endothelial cells from the elastin lamellae results in a tear. The combination of weak media and a tear in the intima leads to rupture of the vessel wall resulting in intramural bleeding. The Pkd1(nl/nl) mouse reveals that polycystin1 is implicated in maintenance of the vessel wall structural integrity, and it is a useful model for dissecting aneurysm formation studies.

Mechanisms Underlying Hypertrophic Remodeling and Increased Stiffness of Mesenteric Resistance Arteries From Aged Rats

The mechanisms associated with structural and mechanical alterations of mesenteric resistance arteries from aged rats were investigated by using pressure myography, confocal microscopy, immunofluorescence, and picrosirius red staining. Arteries from old rats showed: (i) increased wall and media thickness, greater number of smooth muscle cell (SMC) layers but decreased density of SMC; (ii) increased number of adventitial cells; (iii) hypertrophy of nuclei of SMC and endothelial cells; (iv) increased stiffness associated with increased total collagen content and collagen I/III deposition in the media; and (v) similar content but changes in elastin structure in the internal elastic lamina. Hypertrophic outward remodeling in aged rat resistance arteries involve adventitial cells hyperplasia, reorganization of the same number of hypertrophied SMC in more SMC layers leading to thickened media and endothelial cell hypertrophy. Fibrosis associated with collagen deposition and changes in elastin structure might be responsible for the increased stiffness of resistance arteries from aged rats.

Low Smooth Muscle Cell Densities Characterize Sites With Isolated Interstitial Lipid in Coronary Artery Intima

The initial step in the development of atherosclerotic lesions is suggested to be the retention of low-density lipoprotein. This suggestion would seem to imply that some interval of time ought to intervene between the initial retention of interstitial lipid and subsequent influx of fatty streak cellular elements. Yet no evidence for such a waiting interval has been offered. To describe findings from application to coronary arteries from forensic autopsy specimens of a new method for staining lipids in paraffin sections. Isolated interstitial lipid (IIL) (extracellular lipid pools accompanied by no perceptible intracellular lipid or other fatty streak elements at the affected sites) was identified in various intimal compartments, and smooth muscle cell (SMC) numbers were counted at these sites. Isolated interstitial lipid was a surprisingly frequent finding after age 35 years, seen in 87 (84%) of 104 cases. The number of SMCs per unit area of sectioned intima was high in specimens lacking IIL (15.5 SMCs/[100 microm]2), lowest at sites displaying IIL (8.3 SMCs/[100 microm]2), and intermediate at sites lacking IIL in specimens that do contain IIL at some other location (12.6 SMCs/[100 microm]2). These findings plus other evidence support the proposal that retention of lipid most often happens, in aging arteries, at sites where the architecture was previously altered by diluting of constant SMC numbers by expanding collagenous matrix, thereby lowering SMC densities.

Structural and Functional Rearrangement of Great Arterial Vessels in the Late Postresuscitative Period

Objective: to study the specific features of remodeling of the aorta, carotid, and femoral artery in the late postresuscitative period. Materials and methods. Experiments were carried out on non-inbred albino rats when clinical death was stimulated by the method of V. G. Korpachev due to 10-minute cardiovascular fascicle ligation. Before clinical death and 60 days after resuscitation, blood pressure, vascular tissue malonic dialdehyde levels, and plasma biochemiluminescence parameters were recorded and great vascular tissue microscopic and ultramicroscopic studies were conducted. Results. On day 60 following resuscitation, a unitary connective tissue carcass was shown to form in the great vascular intima and media via chaotic synthesis of heretodirection-al fibers. This appeared as elevated levels of collagenous fibers in the intima and media as shown by light microscopy. The aortic, carotid, and femoral arterial media displayed a large number of secreting smooth muscle cells and elevated levels of collagenous fibers, which was an active vascular wall remodeling process. Enhanced free radical processes during reperfusion both in the whole organism and vascular wall tissue were one of the triggers of remodeling processes after ischemia. Impaired vascular tone regulation caused by the development of vascular wall remodeling by the connective tissue vegetation growth type contributed to the development of a hypertensive response in the late period. Conclusion. The late postresuscitative period was marked by increased connective tissue in the media of great vessels, by imperfect recovery of the morphofunc-tional status of the endothelial lining, which was likely to be a cause of increased vascular stiffness and elevated blood pressure in rats 2 months after resuscitation.

Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization

Biodegradable synthetic matrices that resemble the size scale, architecture and mechanical properties of the native extracellular matrix (ECM) can be fabricated through electrospinning. Tubular conduits may also be fabricated with properties appropriate for vascular tissue engineering. Achieving substantial cellular infiltration within the electrospun matrix in vitro remains time consuming and challenging. This difficulty was overcome by electrospraying smooth muscle cells (SMCs) concurrently with electrospinning of a biodegradable, elastomeric poly(ester urethane) urea (PEUU) small-diameter conduit. Constructs were cultured statically or in spinner flasks. Hematoxylin and eosin (H&E) staining demonstrated qualitatively uniform SMCs integration radially and circumferentially within the conduit after initial static culture. In comparison with static culture, samples cultured in spinner flasks indicated 2.4 times more viable cells present from MTT and significantly larger numbers of SMCs spread within the electrospun fiber networks by H&E image analysis. Conduits were strong and flexible with mechanical behaviors that mimicked those of native arteries, including static compliance of 1.6±0.5×10 −3 mmHg −1, dynamic compliance of 8.7±1.8×10 −4 mmHg −1, burst strengths of 1750±220 mmHg, and suture retention. This method to rapidly and efficiently integrate cells into a strong, compliant biodegradable tubular matrix represents a significant achievement as a tissue engineering approach for blood vessel replacement.

Long-Term Continuous Treatment with Sildenafil Ameliorates Aging-Related Erectile Dysfunction and the Underlying Corporal Fibrosis in the Rat1

Aging-related erectile dysfunction is characterized by a loss of smooth muscle cells (SMCs) and fibrosis in the corpora cavernosa, and functionally by corporal veno-occlusive dysfunction (CVOD). Phosphodiesterase 5 (PDE5A) inhibitors, in part via upregulating inducible nitric oxide synthase (NOS2A), have antifibrotic properties in penile tissues. We aimed to determine whether in the aged rat the chronic long-term treatment with sildenafil ameliorates corporal SMC loss and fibrosis, stimulates NOS2A induction, and corrects the associated CVOD. Aged male rats (20 mo old) received sildenafil in their drinking water (20 mg/kg per day) or plain water for 45 days, and untreated young rats (5 mo old) served as controls (n = 8 per group). CVOD was assessed by dynamic infusion cavernosometry (DIC). Collagen:SMC (Masson trichrome) and collagen III:I (picrosirius red) ratios, SMC content (alpha-smooth muscle actin [ACTA2]), cell proliferation (proliferating nuclear antigen [PCNA]), apoptotic death (TUNEL), and NOS2A induction were measured by histochemistry and immunohistochemistry followed by quantitative image analysis. Collagen content was determined by hydroxyproline assay, and transforming growth factor beta-1 (TGFB1); xanthine oxidoreductase (XDH); ACTA2; NOS2A; and the Rho kinase inhibitor protein tyrosine phosphatase, nonreceptor type 11 (PTPN11), and activator, VAV, were measured by quantitative Western blot. In the aged rats treated with sildenafil, the erectile response by DIC was normalized, and the corporal SMC:collagen ratio and SMC number were increased. In addition, sildenafil reduced the corporal collagen content without affecting the collagen III:I ratio, increased the PCNA:apoptosis ratio, and stimulated NOS2A induction, although there was no effect on XDH, TGFB1, PTPN11, or VAV levels. These data show that long-term PDE5A treatment corrected CVOD in the aged rat and partially reversed the aging-related fibrosis and loss of SMC in the corpora cavernosa without affecting TGFB1 or PTPN11 levels, which are markers of oxidative stress. It may be speculated that similar effects may be achieved with this paradigm in men.

Aging-Associated Vascular Phenotype in Mutant Mice With Low Levels of BubR1

Aging is a major risk for stroke and a highly complex biological process believed to involve multiple mechanisms. Mutant mice that express low levels of the spindle assembly checkpoint protein BubR1 are known to develop several aging-associated phenotypes at a very young age, including cataracts, lordokyphosis, loss of subcutaneous fat, and impaired wound healing. However, whether BubR1 acts to prevent vascular aging has not yet been established. The present study was designed to investigate the vascular phenotype of mutant mice with low levels of BubR1. Morphological, functional, and biochemical analyses were performed on aortas and carotid arteries of 3- to 5-month-old BubR1 mutant mice and wild-type littermates. Arterial wall thickness and inner diameter were significantly reduced in BubR1 mutant mice. Arterial walls of BubR1 mutant mice had low numbers of medial smooth muscle cells. Masson trichrome staining showed profound fibrosis in arterial walls of BubR1 mutant. In agreement with these morphological changes, functional analysis of pressurized isolated carotid arteries of BubR1 mutant mice demonstrated reduced elastic properties. Endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to the nitric oxide donor DEA-NONOate were significantly reduced in carotid arteries of BubR1 mutant mice. Furthermore, enzymatic activity of nitric oxide synthase and levels of cyclic GMP were significantly reduced in aortas of mutant mice, but production of superoxide anions was significantly increased. These findings demonstrate that BubR1 insufficiency in mice results in phenotypic changes reminiscent of vascular aging in humans and suggest a role for BubR1 in suppressing the vascular aging process.