Ex vitro experimental study on concentration polari-zation of macromolecules (LDL) at an arterial stenosis

To verify the previous theoretical prediction that the disturbed flow distal to a stenosis enhances lipid accumulation at the blood/arterial wall interface, we designed a canine carotid arterial stenosis model and measured ex vitro the luminal surface concentration of bovine serum albumin (as a tracer mac-romolecule) by directly taking liquid samples from the luminal surface of the artery. The experimental results showed that due to the presence of a filtration flow, the luminal surface albumin concentration cw was higher than the bulk concentration co as predicted by our theory. The measurement revealed that the luminal surface concentration of macromolecules was indeed enhanced significantly in re-gions of the disturbed flow. At Re = 50, the relative luminal surface concentration cw/co was 1.66 ± 0.10 in the vortex region, while the cw/co was 1.37 ± 0.06 in the laminar flow region. When Re increased to 100, the cw/co in the vortex flow region and the laminar flow region reduced to 1.39 ± 0.07 and 1.24 ± 0.04, respectively. The effect of the filtration rate, vw, on the luminal surface concentration of albumin was remarkably apparent. At Re = 50 and 100, when vw = 8.9 ± 1.7 × 10-6 cm/s, cw in the vortex region was 77% and 52% higher than co respectively, meanwhile when vw = 4.8 ± 0.6 × 10-6 cm/s, cw in the vortex region was only 66 % and 39% higher than co respectively. In summary, the present study has provided further experimental evidence that concentration polarization can occur in the arterial system and fluid layer with highly concentrated lipids in the area of flow separation point may be responsible for the formation and development of atherosclerosis.     

Anti-inflammatory and cholesterol-reducing properties of apolipoprotein mimetics: a review

Reduced levels of HDL cholesterol (HDL-C) are a strong independent predictor of coronary artery disease (CAD) risk. The major anti-atherogenic function of HDL is to mediate reverse cholesterol transport. This response is highly dependent on apoA-I and apoE, protein components of HDL. Randomized clinical trials have assessed effects of several classes of drugs on plasma cholesterol levels in CAD patients. Agents including cholestyramine, fibrates, niacin, and statins significantly lower LDL cholesterol (LDL-C) and induce modest increases in HDL-C, but tolerance issues and undesirable side effects are common. Additionally, residual risk may be present in patients with persistently low HDL-C and other complications despite a reduction in LDL-C. These observations have fueled interest in the development of new pharmacotherapies that positively impact circulating lipoproteins. The goal of this review is to discuss the therapeutic potential of synthetic apolipoprotein mimetic peptides. These include apoA-I mimetic peptides that have undergone initial clinical assessment. We also discuss newer apoE mimetics that mediate the clearance of atherogenic lipids from the circulation and possess anti-inflammatory properties. One of these (AEM-28) has recently been given orphan drug status and is undergoing clinical trials.     

Rac3, but not Rac1, promotes ox-LDL induced endothelial dysfunction by downregulating autophagy

The endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis, which can lead to oxidative stress and inflammation. The role of autophagy in the process of atherosclerosis has drawn increasing attention. The human umbilical vein endothelial cells (HUVECs), whose Ras-related C3 botulinum toxin substrate 1 (Rac1) and Rac3 was knockdown, were used to detect whether the possible molecular mechanisms of Rac1 and Rac3 for anti-inflammatory in endothelial cells was effected by downregulation of autophagy. The HUVECs were incubated with ox-LDL. The inflammatory factors and autophagy proteins were evaluated to ascertain and compare the effect of Rac1 and Rac3 on autophagy. Then, 3-methyladenine (3-MA) as an inhibiter of autophagy was used to detect whether the effect of Rac1 and Rac3 was related to autophagy. ox-LDL-induced cell dysfunction in HUVECs was determined by testing the formation of foam cells, the expression of nuclear factor (NF)-κB and nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 and NF-κB p65 and other inflammatory factors, the release of reactive oxygen species by oxidative stress and the dysfunction of the cytomembrane. And ApoE^−/− mice on a high-fat diet were used as an animal model to detect the effect of Rac1 and Rac3 in vivo. The results showed that when Rac1 and Rac3 were decreased in HUVECs, the cell dysfunction caused by ox-LDL was inhibited. If 3-MA was used to inhibit autophagy in Rac1 and Rac3 knockdown cells, the injury induced by ox-LDL on the cells was recovered. These results indicated that the effect of Rac1 and Rac3 was combined with ox-LDL, which was related to inhibition of autophagy. The effect of Rac3 was more significant than that of Rac1.     

Lipid-modified proteins as biomarkers for cardiovascular disease: a review

Lipid-modified proteins are classified based on the identity of the attached lipid, a post- or co-translational modification required for their biological function. At least five different lipid modifications of cysteines, glycines and other residues on the COOH- and NH₂-terminal domains have been described. Cysteine residues may be modified by the addition of a 16-carbon saturated fatty acyl group by a labile thioester bond (palmitoylation) or by prenylation processes that catalyze the formation of thioether bond with mevalonate derived isoprenoids, farnesol and geranylgeraniol. The NH₂-terminal glycine residues may undergo a quite distinct process involving the formation of an amide bond with a 14-carbon saturated acyl group (myristoylation), while glycine residues in the COOH-terminal may be covalently attached with a cholesterol moiety by an ester bond. Finally, cell surface proteins can be anchored to the membrane through the addition of glycosylphosphatidylinositol moiety. Several lines of evidence suggest that lipid-modified proteins are directly involved in different steps of the development of lesions of atherosclerosis, from leukocyte recruitment to plaque rupture, and their expression or lipid modification are likely altered during atherogenesis. This review will briefly summarize the different enzymatic pathways of lipid modification and propose a series of lipid-modified proteins that can be used as biomarkers for cardiovascular disease.     

The effect of coculture with human smooth muscle cells on the proliferation,the IL‐1 β secretion,the PDGF production and tube formation of human aortic endothelial cells

Endothelial cells (ECs) and smooth muscle cells (SMCs), which are the major component cells of blood vessels, produce various bioactive substances and communicate with each other through them. Although several studies of the interaction between ECs and SMCs have been reported, the effect of coculture with SMCs on ECs is still obscure. To clarify the interaction of ECs and SMCs, we examined the effect of coculture with SMCs on the proliferation, the IL‐1β secretion, the PDGF production and tube formation of ECs, using the coculture model: transferable wells and collagen gel. IL‐1 and PDGF are considered to be related to progression of atherosclerosis. Proliferation and tube formation of ECs are associated with repair of vessels. In the transferable well system coculture with SMCs stimulated the proliferation of ECs, and enhanced the IL‐1β secretion of ECs and in the collagen gel system coculture with SMCs induced the tube formation of ECs, and appeared to enhance the PDGF production of ECs. In conclusion, the effect of coculture with SMCs on ECs has two conflicting aspects: progression of atherosclerosis and angiogenesis. These results suggest that an imbalance of their effects may lead to pathological events. Copyright © 1999 John Wiley & Sons, Ltd.     

血管再生中的内皮祖细胞

循环血液里存在一种被称为内皮祖细胞(endothelial progenitor cells,EPCs)的祖细胞亚群,具有在体内外分化为成熟内皮细胞的能力。根据内皮祖细胞与其他血液细胞的粘附能力的差异和内皮祖细胞的抗原特异性,内皮祖细胞可通过贴壁培养和免疫磁珠筛选而分离获得。内皮祖细胞可特异性表达三种祖细胞分子标志：CD133、CD34和血管内皮生长因子受体-2。当内皮祖细胞分化为成熟内皮细胞后,血小板内皮细胞粘附分子-1(CD31)、血管内皮粘附素(VE-cadherin,又称CD144)和Ⅷ因子(vWF)表达将上调。越来越多的证据显示,内皮祖细胞有利于体内内皮损伤后修复和血管再生。我们的研究发现,内皮祖细胞可修复apoE-缺陷小鼠血管移植物中的损伤内皮并且在动脉血管外膜中存在大量的血管祖细胞。然而,在机体的血管再生和动脉硬化的形成进程中,这些内皮祖细胞的作用和机制还不太明确。另外,有关机体内相应心血管疾病危险因素是如何影响内皮祖细胞功能的机制也不清楚。因此,对内皮祖细胞的归巢、释放和粘附机制的进一步深入研究将有助于人们探索内皮祖细胞的基础理论和临床应用价值。     

H2O2 down-regulates SIRT7’s protective role of endothelial premature dysfunction via microRNA-335-5p

Endothelial senescence is believed to constitute the initial pathogenesis of the atherosclerotic cardiovascular disease (ASCVD). MicroRNA-335-5p (miR-335-5p) expression is significantly up-regulated in oxidative stress-induced endothelial cells (ECs). Sirtuin7 (SIRT7) is considered to prevent EC senescence, yet data on its response to ASCVD risk factors are limited. The present study analyzed the elevated levels of miR-335-5p and the decreased levels of SIRT7 in human umbilical vein endothelial cells (HUVECs), and found that high glucose, tumor necrosis factor-α (TNF-α), and H₂O₂ are the three contributing factors that induced cellular senescence. The current study also assessed premature endothelial senescence and decreased proliferation, adhesion, migration, and nitric oxide (NO) secretion in HUVECs with these risk factors together with SIRT7–siRNA transfection. It found that the miR-335-5p inhibitor attenuated the down-regulation of SIRT7 expression induced by oxidative stress in HUVECs, and SIRT7 overexpression exerts a rescue effect against miR-335-5p-induced endothelial dysfunction. Furthermore, the direct binding of miR-335-5p to SIRT7 was observed in human embryonic kidney cells 293T (HEK 293T). Therefore, it can be inferred that miR-335-5p down-regulates the expression of SIRT7 in human cells. Current findings may provide deeper insights into the underlying mechanisms of endothelial senescence and potential therapeutic targets of ASCVD as well as other age-related diseases.     

Extracellular Nucleotide Catabolism in Aortoiliac Bifurcation of Atherosclerotic ApoE/LDLr Double Knock Out Mice

Atherosclerosis is a consequence of diverse pathologies that could be affected by signaling mediated by nucleotides and their metabolites. Concentration of specific nucleotide derivatives in the proximity of purinergic receptors is controlled by extracellular enzymes such as ecto-nucleoside triphopsphate diphosphohydrolase (eNTPD), ecto-5′-nucleotidase (e5NT), and ecto-adenosine deaminase (eADA). To estimate changes in metabolism of extracellular nucleotides in the atherosclerotic vessel wall, aortoiliac bifurcation of ApoE/LDLr (–/–) mice was perfused with solution containing adenosine-5′-triphosphate (ATP), adenosine-5′-monophosphate (AMP) or adenosine. Formation of the product of eNTPD, e5NT or eADA was measured by high performance liquid chromatography (HPLC). The most significant difference between ApoE/LDLr (–/–) and wild-type mice was several times higher rate of conversion of adenosine to inosine catalyzed by eADA activity. This highlights potential decrease in intravascular adenosine concentration in atherosclerosis.     

The Roles of Protein Kinase C βI and βII in Vascular Smooth Muscle Cell Proliferation

The role of protein kinase C (PKC) on proliferation of A10 vascular smooth muscle cells (VSMC) was studied by overexpressing specific PKC-βI and -βII isozymes. PKC-βI and -βII are derived from alternative splicing of the exon encoding the carboxy-terminal (C-terminal) 50 or 52 amino acids, respectively. The differential functions of the two isozymes with regard to cell proliferation, DNA synthesis, and the cell cycle were investigated in A10 cells, a clonal cell line of VSMC from rat aorta, and in A10 cells overexpressing PKC-βI and PKC-βII (βI-A10 and βII-A10). PKC levels were increased three- to fourfold in heterogeneous cultures of stably transfected cells. Although doubling time of A10 cells was 36 h, the cell doubling time in βI-A10 cells decreased by 12 h, and, in contrast, the doubling time of βII-A10 cells increased by 12 h compared to A10 cells. The increase of [³H]thymidine (TdR) incorporation was accelerated and increased in βI-A10 cells, but slowed and diminished in βII-A10 cells compared to A10 and control cells transfected with empty vector. Cell cycle analysis of βI-A10 cells showed an acceleration of S phase entry while βII-A10 cells slowed S phase entry. These results suggest that PKC-βI and PKC-βII regulate cell proliferation bidirectionally and that PKC-βI and PKC-βII may have distinct and opposing functions as cell cycle check point mediators during late G₁phase and may regulate S phase entry in A10 VSMC.     

Coexistence of substance P,neuropeptide Y,VIP, and CGRP in the nerve fibers of the carotid labyrinth of the bullfrog,Rana catesbeiana: a double-labelling immunofluorescence study in combination with alternate consecutive sections

Double immunohistochemical staining with rhodamine- and fluorescein isothiocyanate (FITC)-conjugated antisera revealed the coexistence of substance P (SP) and neuropeptide Y (NPY), and SP and calcitonin gene-related peptide (CGRP) in most nerve fibers in the intervascular stroma of the carotid labyrinth of the bull-frog, Rana catesbeiana, although there were a few fibers which showed only SP- or NPY-immunoreactivity. Approximately one third of SP-immunoreactive fibers also showed coexistence with vasoactive intestinal polypeptide (VIP)-immunoreactivity, and a few fibers contained VIP without SP. The combination of the double immunofluorescence technique and alternate consecutive sections further demonstrated the possible coexistence of SP, VIP, NPY, and CGRP. This coexistence of four different peptides in the same nerve fibers was proved by the following two evident facts: 1) some SP fibers which demonstrated coexistence with NPY-immunoreactivity were assumed to be continuous with those showing VIP-immunoreactivity, and 2) almost all of the SP fibers showed coexistence with CGRP-immunoreactivity. By this reasoning, nearly one third of SP fibers may demonstrate coexistence with NPY-, VIP-, and CGRP-immunoreactivities. These multiple peptides might be involved in vascular regulatory function, which is a possible function of the amphibian carotid labyrinth.