Contribution of Systemic Inflammation to the Pathogenesis of Endothelial Dysfunction

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.

The effect of 24 h exposure to ethanol at concentrations of 50 and 100 mM on the proliferative, migration, and contractile activity of thoracic aortic smooth muscle cells of rats of three age groups was studied, the early neonatal period of development (1-week-old rats), puberty (5-month-old rats), and the initial period of aging (one-year-old rats). Real-time PCR was used to evaluate the proliferative activity of smooth muscle cells by comparing the relative amount of genomic DNA between control and experiment; wound healing assay was used to determine the migration activity of smooth muscle cells; and a method for assessing the degree of compression of collagen gel was used to determine the level of contractile activity of cells. Ethanol at a concentration of 50 mM did not change the studied properties of aortic smooth muscle cells of rats of all age groups. In the presence of ethanol at a concentration of 100 mM, the following effects were observed: an increase of ~38% (p < 0.01) in proliferative and a decrease of ~12% (p < 0.01) in contractile activity of smooth muscle cells of the aorta of rats of the early neonatal period, as well as a decrease in contractile activity (by ~7%, p < 0.01) and proliferative activity (by ~21%, p < 0.01) of cells of 5-month-old and one-year-old rats. It can be concluded that ethanol at a concentration of 100 mM has different effects on the studied rat cells depending on the age group of animals, and the smooth muscle cells of the aorta of rats in the early neonatal period of development are more sensitive to the effects of ethanol.

Structure–function relationships of citrus limonoids on p38 MAP kinase activity in human aortic smooth muscle cells

13.12 - Oxidants and Endothelial Dysfunction

Oxidants and Endothelial Dysfunction

Rabbit smooth muscle cells (SMC) express types I and II scavenger receptors (ScR) that are up-regulated by platelet secretion products. In the current studies we investigated the effect of growth factors secreted by platelets on ScR activity in rabbit and human SMC. Platelet-derived growth factor (PDGF BB) and transforming growth factor beta 1 (TGF-beta 1) at 10 ng/ml increased ScR activity in rabbit SMC (by approximately 4- and 2-fold, respectively) but not in human SMC. Epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) alone had little effect on SMC ScR activity. The growth factors had synergistic effects on ScR activity and on types I and II ScR mRNA expression. In rabbit SMC, PDGF BB, EGF, and TGF-beta 1 together stimulated ScR activity 12-fold. In human SMC, EGF and TGF-beta 1, together with either IGF-I or PDGF BB, stimulated receptor activity approximately 7-fold. Growth factor-mediated induction of ScR activity in rabbit and human SMC was blocked by the tyrosine kinase inhibitor tyrphostin 47, whereas the induction of ScR activity in rabbit but not human SMC was blocked by the protein kinase C inhibitor MDL.29,152. Studies using neutralizing antibodies demonstrated that TGF-beta 1 is the predominant factor in in vitro preparations of platelet secretory products which regulates ScR activity. The growth factors that act synergistically in regulating ScR activity in vitro are all present in atherosclerotic lesions, where they are produced by macrophages, endothelial cells, SMC, and platelets. The data suggest that these growth factors may regulate ScR activity in SMC in vivo and contribute to foam cell formation.

During infection or inflammation, cells of the blood vessel wall, such as endothelial cells (EC) and smooth muscle cells (SMC), contribute to the regulation of the immune response by production of cytokines or expression of adhesion molecules. Little is known about the mechanism(s) involved in the stimulation of vascular cells by endotoxin (lipopolysaccharide [LPS]). As reported previously, LPS antagonists reduce LPS-induced cytokine production or adhesion in vitro specifically, suggesting a specific LPS recognition mechanism. We thus investigated the role of CD14 for stimulation of vascular SMC by LPS. Complement-fixing antibodies directed against CD14 (LeuM3, RoMo I, or Mo2) lysed monocytes but failed to mediate lysis of EC or SMC, indicating the lack of endogenous membrane CD14 in vascular cells. In addition, we did not detect expression of CD14 protein on EC and SMC in cell sorting analysis or cell immunoassay experiments. These observations are in line with our finding that a CD14 probe did not hybridize with mRNA or EC or SMC in Northern (RNA) blot experiments, although it hybridized well with monocyte-derived mRNA. We obtained the same results with the much more sensitive reverse transcription-PCR. Since the vascular SMC did not express endogenous CD14, we investigated the role of human serum-derived soluble CD14 (sCD14) for activation of SMC by LPS. In medium containing human serum, anti-CD14 antibodies inhibited activation of SMC by LPS. In contrast, the same antibodies did not inhibit activation of cells cultured in medium containing fetal calf serum. SMC cultured in sCD14-depleted medium responded 1,000-fold less to LPS than cells cultured in presence of sCD14. Reconstitution of sCD14-depleted serum or supplementation of serum-free medium with recombinant CD14 restored the capacity of the cells to respond to LPS. These results show that specific activation of vascular SMC by LPS does not involve binding to endogenous membrane CD14, but that the activation of vascular SMC by LPS is mediated to a great extent by serum-derived sCD14.

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle.

Chapter 14 - Association of phospholipase C and D in cardiovascular risk prediction and progression

Cardiovascular diseases have been the leading cause of death worldwide for a long time. Despite numerous studies on the pathogenetic mechanisms of cardiovascular diseases, there are many debatable issues. In recent years, an increasing number of scientific reports have appeared regarding the presence of common mechanisms in the deve­lopment of bone tissue and arterial calcification. One of the connecting links in this interaction is considered to be the impact of osteocalcin. Osteocalcin is a vitamin K-dependent protein of the bone matrix, synthesized by osteoblasts. The main function of osteocalcin is the synthesis of hydroxyapatites as main mineral component of bone tissue. In addition, osteocalcin has a wide range of extraosseous effects, the most studied is participation in the regulation of glycolipid and ener­gy metabolism. Research on the osteocalcin role in the development and progression of cardiovascular diseases are scarce, the available data is contradictory. For a deeper understanding of this problem, we conducted a systematic analysis of modern literature based on data from the scientific databases Medline (Pubmed), Scopus, Web of Science, Google Scholar, and Cochrane Library for 2013–2023. According to its results, osteocalcin is a potential biomarker of cardiovascular status, its increased values are associated with a potentially protective mechanism against the development of cardiovascular diseases. Contradictory views on the understanding of the pathogenetic mechanism of influence of general osteocalcin and its forms on the course of cardiovascular diseases necessitate conduction of further research.

Sex Differences in Hypertension: Contribution of the Renin–Angiotensin System

Clinical and epidemiological data show that biological sex is one of the major determinants for the development and progression of cardiovascular disease (CVD). Impaired endothelial function, characterized by an imbalance in endothelial Nitric Oxide Synthase (eNOS) activity, precedes and accelerates the development of CVD. However, whether there is any sexual dimorphism in eNOS activity and function in endothelial cells (ECs) is still unknown. Here, by independently studying human male and female ECs, we found that female ECs expressed higher eNOS mRNA and protein levels both in vitro and ex vivo. The increased eNOS expression was associated to higher enzymatic activity and nitric oxide production. Pharmacological and genetic inhibition of eNOS affected migratory properties only in female ECs. In vitro angiogenesis experiments confirmed that sprouting mostly relied on eNOS-dependent migration in female ECs. At variance, capillary outgrowth from male ECs was independent of eNOS activity but required cell proliferation. In this study, we found sex-specific differences in the EC expression, activity, and function of eNOS. This intrinsic sexual dimorphism of ECs should be further evaluated to achieve more effective and precise strategies for the prevention and therapy of diseases associated to an impaired endothelial function such as CVD and pathological angiogenesis.

We demonstrated previously that maternal nutrient restriction during pregnancy impairs an EDHF‐like pathway that is mediated via BKCa activation in fetal coronary arteries (CA). We now show that the impairment is not likely due to altered BKCa activity in CA smooth muscle cells. Pregnant ewes were fed a control or nutrient restricted diet from day 50–130 of gestation. In CA isolated from control fetal lambs, relaxation to bradykinin was partially inhibited by nitro‐l‐arginine (NLA). Iberiotoxin nearly abolished the NLA‐resistant response to bradykinin. In CA from undernourished animals, relaxation to bradykinin was fully suppressed by NLA. Whole‐cell BKCa currents were nearly identical in CA smooth muscle cells from control and nutrient restricted animals (peak current density= 46 ± 5 vs. 39 ± 6 pA/pF; n=6). 14,15‐EET (putative EDHF in CA) caused fetal CA relaxation and BKCa activation that was unaffected by maternal nutrient restriction. Similar results were obtained with the BKCa‐openers, BMS 191011 and NS 1619. Relaxation to bradykinin in fetal CA is mediated by NO, and by an EDHF‐like pathway that involves BKCa activation. Maternal undernutrition during pregnancy results in loss of the EDHF‐like pathway in fetal CA, an effect that is likely due to endothelial dysfunction (i.e. impaired synthesis and/or release of an EDHF‐like mediator) rather than impaired BKCa activity in CA smooth muscle cells. (NIH HD61532)

Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture

Platelets, monocytes, and endothelial cells are instrumental in the development and progression of cardiovascular diseases. Inflammation, a key process underlying cardiovascular disorders, is accompanied and amplified by activation of platelets and consequent binding of such platelets to the endothelium. There, platelet-derived chemokines, in conjunction with increased expression of adhesion molecules, promote the recruitment of circulating monocytes that will eventually migrate across the endothelial lining of the vessel into the tissues. Additionally, platelets may already become activated in the circulation and may form platelet-monocyte complexes, which show increased adhesive and migratory capacities themselves but also facilitate recruitment of noncomplexed leukocytes. They should therefore be considered as important mediators of inflammation. In molecular terms, these events are additionally governed by chemokines released and presented by the endothelium as well as the different classes of endothelial adhesion molecules that regulate the interactions among the various cell types. Most important in this respect are the selectins and their ligands, such as P-selectin glycoprotein (GP) ligand 1, and the integrins binding to Ig-like cell adhesion molecules as well as to GP, such as von Willebrand factor, present in the extracellular matrix or on activated endothelium. This review aims to provide an overview of these complex interactions and of their functional implications for inflammation and development of cardiovascular disease.