Coating Decellularized Equine Carotid Arteries with CCN1 Improves Cellular Repopulation, Local Biocompatibility, and Immune Response in Sheep

Blood vessels are permanently subjected to mechanical forces in the form of stretch, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow and shear stress. Significant variations in mechanical forces, of physiological or physiopathological nature, occur in vivo. These are accompanied by phenotypical modulation of smooth muscle cells and endothelial cells, producing structural modifications of the arterial wall. In all the cases, vascular remodelling can be allotted to a modification of the tensional strain or shear, and underlie a trend to reestablish baseline mechanical conditions. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by pressure and shear stress. The cytoskeleton and other structural components have an established role in mechanotransduction, being able to transmit and modulate tension within the cell via focal adhesion sites, integrins, cellular junctions and the extracellular matrix. Mechanical forces also initiate complex signal transduction cascades, including nuclear factor-kappaB and mitogen-activated protein kinase pathways, leading to functional changes within the cell.

In this study, we compared the effect of the essential oil of Citrus bergamia Risso [bergamot, bergamot essential oil (BEO)] on the intracellular Ca levels in vascular endothelial (EA) and mouse vascular smooth muscle (MOVAS) cells, using the fura-2 fluorescence technique. BEO caused an initial transient increase in intracellular Ca concentration ([Ca]i) in EA cells, followed by a decrease, whereas it induced a sustained increase in [Ca]i in MOVAS cells. Linalyl acetate (LA) as a major component of BEO-induced [Ca]i mobilization was similar to BEO in EA cells. The increase of [Ca]i by LA was higher in EA cells than in MOVAS cells. [Ca]i rise induced by extracellular Ca application was significantly blocked by BEO or LA in EA cells but not in MOVAS cells, suggesting that BEO and LA block Ca influx in EA cells. The present results suggest that BEO and LA differentially modulate intracellular Ca levels in vascular endothelial and smooth muscle cells. In addition, blockade of Ca influx by BEO and LA in EA cells may explain the protective effects of BEO on endothelial dysfunction associated with cardiovascular disease.

Cultured bovine aortic smooth muscle (BASM) and endothelial (BAE) cells, like articular chondrocytes, synthesize two populations of small chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) of similar size as PG-I/biglycan and PG-II/decorin. However, Northern blot analyses demonstrated that BAE cells express detectable amounts of mRNA transcripts only for PG-I/biglycan, whereas BASM cells and articular chondrocytes express mRNA transcripts for both PG-I/biglycan and PG-II/decorin. Endothelial cells from human umbilical vein also expressed detectable amounts of mRNA transcripts only for PG-I/biglycan, and not PG-II/decorin. Antiserum raised against bovine PG-II/decorin immunoprecipitated an apparent single PG species with relative molecular mass (Mr) of approximately 120,000-180,000 from BASM cell and articular chondrocyte cultures but failed to immunoprecipitate an equivalent PG species from BAE cell cultures, consistent with the results from Northern blot analysis. In contrast, immunoprecipitations by antisera to PG-I/biglycan indicated that cultured endothelial cells synthesize two forms of PG-I/biglycan with Mr values slightly larger than 200,000 and 120,000-140,000. It is likely, based on the magnitude of the size difference, that these two forms of PG-I/biglycan differ in the number of glycosaminoglycan chains. Additionally, BASM but not BAE cells were found to express detectable amounts of mRNA transcripts for type I collagen. The above results indicate that the two main cell types of the vascular wall, endothelial and smooth muscle cells, express different sets of small interstitial CS/DS PGs and that the synthesis of PG-II/decorin by these cells correlates with the expression of type I collagen, a collagen known to interact specifically with this PG. These differences in the expression of extracellular matrix molecules may be important in regulating the cell type-specific functions of endothelial and smooth muscle cells within the vascular tissue.

endothelial progenitor cells (EPCs) are a unique cell type found circulating in the peripheral blood with the capacity to become mature endothelial cells. EPCs can be released from many sources including the bone marrow, adipose tissue, the vessel wall, as well as potentially the spleen, liver, and

The FGF-2-Derived Peptide FREG Inhibits Melanoma Growth In Vitro and In Vivo

Distribution of receptors mediating phosphoinositide hydrolysis in cultured human umbilical artery smooth muscle and endothelial cells

The use of self-assembled monolayers (SAMs) as a polymer-free platform to deliver an antiproliferative drug, paclitaxel (PAT), from a stent material cobalt-chromium (CoCr) alloy has been previously demonstrated. In this study, the interaction of human aortic endothelial cells (ECs) and human aortic smooth muscle cells (SMCs) with CoCr alloy surfaces coated with SAMs- (SAMs-CoCr) and PAT-deposited SAMs (PAT-SAMs-CoCr) was investigated. A polished CoCr with no coatings was used as a control. The viability, proliferation, morphology, and phenotype of ECs and SMCs were investigated on these samples. SAMs-CoCr significantly enhanced the growth of ECs. Also, the ECs were well spreading with its typical morphological features and showed stronger PECAM-1 expression on SAMs-CoCr. This showed that the SAMs-CoCr surface is conducive to endothelialization. For PAT-SAMs-CoCr, although the adhesion of ECs was lower, the cells continued to proliferate with some degree of spreading and limited PECAM-1 expression. For SMCs, a significant decrease in the cell proliferation was observed on SAMs-CoCr when compared with that of Control-CoCr. PAT-SAMs-CoCr showed maximum inhibitory effect on the proliferation of SMCs. Also, the SMCs on PAT-SAMs-CoCr displayed a poorly spread discoid morphology with disarranged α-actin filaments. This showed that the PAT released from the SAMs platform successfully inhibited the growth of SMCs. Thus, this study showed the interaction of ECs and SMCs with SAMs-CoCr and PAT-SAMs-CoCr for potential uses in stents and other cardiovascular medical devices.

Inhibitory effect of type 1 collagen gel containing α-elastin on proliferation and migration of vascular smooth muscle and endothelial cells

Primary cultures of rat vascular endothelial and smooth muscle cells were developed as models to study xenobiotic-induced cytotoxicity. Endothelial and smooth muscle cells were isolated by enzymatic digestion and mechanical dissociation of rat thoracic aortae. Optimal cell growth and minimal fibroblast contamination in cultures of both cell types were obtained in Medium 199 supplemented with 10% fetal bovine serum. Cultured cells were characterized by distinctive morphologic features and growth patterns. Intercellular endothelial cell junctions were selectively stained with silver nitrate. Endothelial cells also exhibited a nonthrombogenic surface, as reflected by platelet-binding studies. Confluent cultures of smooth muscle cells, but not endothelial cells, contracted in response to norepinephrine (10 microM). Cultures of both cell types were exposed to acrolein (2, 5 or 50 ppm), an environmental pollutant, for 4 and 24 h. Morphologic damage, lactate dehydrogenase release, and cellular thiol content were used as indices of cytotoxicity. Acrolein-induced enzyme leakage and morphologic alterations were dose- and time-dependent and more pronounced in cultures of smooth muscle cells than in endothelial cells. The total thiol content of endothelial cells exposed to acrolein (50 ppm) for 24 h was not significantly different from that of respective controls. In contrast, the content of treated smooth muscle cells was higher than that of controls. These observations show that primary cultures of vascular cells provide a useful model to evaluate xenobiotic-induced cytotoxicity. The information obtained using a cell culture system may be complemented by the use of other in vivo and in vitro models to determine the mechanisms by which xenobiotics cause vascular cell injury.

Calcium dynamics and intercellular communication in arterial smooth muscle cells

In the lesions of atherosclerosis, vascular smooth muscle cells (SMC) display many functions characteristic of cytokine activation that likely contribute importantly to ongoing inflammation during human atherogenesis. The transcription factor nuclear factor kappa-B (NFkappaB) often mediates the effects of cytokines on target cells, but the identity of Rel family members important in human SMC activation remains uncertain. In vitro, human SMC express multiple Rel family members. Of these, dimers of p65 and p50, but not a putative SMC-Rel, comprise basal and inducible NFkappaB binding activities. SMC express two inhibitor proteins IkappaBbeta and IkappaBalpha. Interleukin-1beta stimulation caused transient loss of IkappaBalpha and a sustained decrease of IkappaBbeta that correlated with increased and persistent levels of p65/p50 protein and binding activity in the nucleus. SMC cultured under serum-free conditions displayed little NFkappaB activity, but addition of serum or platelet-derived growth factor did activate NFkappaB. In situ analyses showed no evidence for basal NFkappaB activity in SMC in vivo as nonatherosclerotic arteries did not contain nuclear p65 or p50 protein. However, the nuclei of intimal SMC within human atheroma did contain both Rel proteins. We conclude that (i) dimers of p65 and p50, but not SMC-Rel, comprise NFkappaB complexes in human SMC; (ii) stimulatory components in serum activate NFkappaB and likely account for previously reported "constitutive" NFkappaB activity in cultured SMC; and (iii) exposure to inflammatory cytokines may produce prolonged NFkappaB activation in SMC because of sustained decreases in the inhibitory subunit IkappaB-beta.

Prevention of stenosis after vascular reconstruction: Pharmacologic control of intimal hyperplasia — A review

Pulmonary arterial hypertension (PAH) is a syndrome in which pulmonary arterial obstruction increases pulmonary vascular resistance, which leads to right ventricular (RV) failure and a 15% annual mortality rate. The present review highlights recent advances in the basic science of PAH. New concepts clarify the nature of PAH and provide molecular blueprints that explain how PAH is initiated and maintained. Five basic science concepts provide a framework to understand and treat PAH: (1) Endothelial dysfunction creates an imbalance that favors vasoconstriction, thrombosis, and mitogenesis. Restoration of this balance by inhibition of endothelin and thromboxane or augmentation of nitric oxide (NO) and prostacyclin is the paradigm on which most current therapy is based. (2) PAH has a genetic component. Mutations (bone morphogenetic protein receptor-2 [BMPR2]) and single-nucleotide polymorphisms (SNPs; ion channels and transporter genes) predispose to PAH. (3) Excess proliferation, impaired apoptosis, and glycolytic metabolism in pulmonary artery smooth muscle, fibroblasts, and endothelial cells suggest analogies to cancer. Many experimental therapies reduce PAH by decreasing the proliferation/apoptosis ratio; these include inhibitors of pyruvate dehydrogenase kinase (PDK), serotonin transporters (SERT), survivin, 3-hydroxy-3-methylglutaryl coenzyme A reductase, transcription factors (hypoxia-inducible factor [HIF]-1α and nuclear factor of activated T lymphocytes [NFAT]), and tyrosine kinases. Augmentation of voltage-gated K+ channels (Kv1.5) and BMPR2 signaling also addresses this imbalance. Tyrosine kinase inhibitors used to treat cancer are currently in phase 1 PAH trials. (4) Refractory vasoconstriction may occur due to rho kinase activation. Fewer than 20% of PAH patients respond to conventional vasodilators; however, refractory vasoconstriction may respond to rho kinase inhibitors. (5) The RV can be targeted therapeutically. Although increased afterload initiates RV failure, which is the major cause of death/dysfunction in PAH, the RV may be amenable to cardiac-targeted therapies. The RV in PAH has features of ischemic, hibernating myocardium. Guided by these new …

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1 beta (IL-1 beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1 beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1 beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1 beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1 beta to see if IL-1 beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1 beta, and IL-1 beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1 beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1 beta induces VEGF gene expression in smooth muscle cells. This IL-1 beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.

The inflammatory etiology of atherosclerosis has prompted a search for biomarkers of inflammation that predict risk for coronary artery disease and its sequelae. Within the acute coronary syndromes (ACS), inflammatory biomarkers may provide independent information regarding pathophysiology, prognosis, and optimal therapeutic strategies. On the basis of the hypothesis that different pathophysiological processes provide nonoverlapping information regarding risk stratification and disease management, this review series addresses biomarkers for each step in the inflammatory process that leads to ACS. Part I reviewed cytokines; this part reviews acute-phase reactants and biomarkers of endothelial cell activation; subsequent parts will address biomarkers of oxidative stress, angiogenesis, extracellular matrix degradation, and platelet activation. In the acute-phase response, cytokines drive production of acute-phase reactants, defined by >25% change in circulating concentration during an inflammatory response. These markers may also remain elevated chronically because of continuing inflammatory stimuli (Table). View this table: Inflammatory Biomarkers in ACS ### C-Reactive Protein C-reactive protein (CRP) is a pentraxin acute-phase protein, members of which are evolutionarily conserved in most vertebrates.1 Hepatocytes and possibly smooth muscle cells and macrophages transcriptionally activate production of CRP in response to inflammatory cytokines, including interleukin-1 and interleukin-6.2 CRP is a robust clinical marker because of its stability, reproducible results, and ease of assay. Although it was originally proposed as a nonspecific marker of inflammation, several reports suggest that CRP may play a direct pathophysiological role in the development and progression of atherosclerosis. Proposed mechanisms include induction of endothelial dysfunction,3 promotion of foam cell formation,4 inhibition of endothelial progenitor cell survival and differentiation,5 and activation of complement in atherosclerotic plaque intima6 and ischemic myocardium.7 Patients with ACS have elevations in CRP in association with their presenting symptoms. There appears to be a bimodal CRP response among patients with ACS. In some patients, CRP may remain elevated for …