Search

Objective: Arteriogenesis, describing the process of collateral artery growth, is activated by fluid shear stress (FSS). Since this vascular mechanotransduction may involve microRNAs (miRNAs), we investigated the FSS-induced expression of vascular cell miRNAs and their functional impact on collateral artery growth during arteriogenesis. Approach and Results: To this end, rats underwent femoral artery ligation and arteriovenous anastomosis to increase collateral blood flow to maximize FSS and trigger collateral vessel remodeling. Five days after surgery, a miRNA expression profile was obtained from collateral tissue, and upregulation of 4 miRNAs (miR-24-3p, miR-143-3p, miR-146a-5p, and miR-195-5p) was verified by quantitative polymerase chain reaction. Knockdown of miRNAs at the same time of the surgery in an in vivo mouse ligation and recovery model demonstrated that inhibition of miR-143-3p only severely impaired blood flow recovery due to decreased arteriogenesis. In situ hybridization revealed distinct localization of miR-143-3p in the vessel wall of growing collateral arteries predominantly in smooth muscle cells. To investigate the mechanotransduction of FSS leading to the increased miR-143-3p expression, cultured endothelial cells were exposed to FSS. This provoked the expression and release of TGF-β (transforming growth factor-β), which increased the expression of miR-143-3p in smooth muscle cells in the presence of SRF (serum response factor) and myocardin. COL5A2 (collagen type V-α2)-a target gene of miR-143-3p predicted by in silico analysis-was found to be downregulated in growing collaterals., Conclusions: These results indicate that the increased miR-143-3p expression in response to FSS might contribute to the reorganization of the extracellular matrix, which is important for vascular remodeling processes, by inhibiting collagen V-α2 biosynthesis.

Objective: Restoration of patency is a natural target of vascular remodeling after venous thrombosis that involves vascular endothelial cells and smooth muscle cells, as well as leukocytes. Acute pulmonary emboli usually resolve <6 months. However, in some instances, thrombi transform into fibrous vascular obstructions, resulting in occlusion of the deep veins, or in chronic thromboembolic pulmonary hypertension (CTEPH). We proposed that dysregulated thrombus angiogenesis may contribute to thrombus persistence., Approach and Results: Mice with an endothelial cell-specific conditional deletion of vascular endothelial growth factor receptor 2/kinase insert domain protein receptor were used in a model of stagnant flow venous thrombosis closely resembling human deep vein thrombosis. Biochemical and functional analyses were performed on pulmonary endarterectomy specimens from patients with CTEPH, a human model of nonresolving venous thromboembolism. Endothelial cell-specific deletion of kinase insert domain protein receptor and subsequent ablation of thrombus vascularization delayed thrombus resolution. In accordance with these findings, organized human CTEPH thrombi were largely devoid of vascular structures. Several vessel-specific genes, such as kinase insert domain protein receptor, vascular endothelial cadherin, and podoplanin, were expressed at lower levels in white CTEPH thrombi than in organizing deep vein thrombi and organizing thrombi from aortic aneurysms. In addition, red CTEPH thrombi attenuated the angiogenic response induced by vascular endothelial growth factor., Conclusions: In the present work, we propose a mechanism of thrombus nonresolution demonstrating that endothelial cell-specific deletion of kinase insert domain protein receptor abates thrombus vessel formation, misguiding thrombus resolution. Medical conditions associated with the development of CTEPH may be compromising early thrombus angiogenesis.

Objective: The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) binds a number of ligands and counter-receptors and thereby is a major determinant in regulation of leukocyte adhesion and extravasation. Vitronectin (VN) is an adhesion-promoting factor that is abundantly present as matrix molecule in vascular diseases such as atherosclerosis. Until now, only an indirect interaction between Mac-1 and VN via the urokinase receptor (urokinase plasminogen activator receptor) was known. We now propose that Mac-1 and VN can directly interact with each other., Methods and Results: In an in vitro system with purified components, Mac-1 specifically bound the multimeric matrix form of VN but not the monomeric plasma form. Using various competitors, the interaction domains in Mac-1 and VN were localized. Mac-1-expressing but not untransfected Chinese hamster ovary cells adhered strongly on VN. Introduction of a GFFKR deletion in the alphaM subunit of Mac-1, which increases the constitutive activation of the integrin, led to increased adhesion on VN. Peripheral human blood neutrophils adhered and migrated on multimeric VN in a Mac-1-dependent manner., Conclusions: These results show that there is a specific integrin-affinity-regulated interaction between Mac-1 and the matrix form but not the plasma form of VN that may significantly participate in leukocyte adhesion and extravasation.

In 1997, investigators isolated a secreted glycoprotein that blocked osteoclast differentiation from precursor cells, prevented osteoporosis (decreased bone mass) when administered to ovariectomized rats, and resulted in osteopetrosis (increased bone mass) when overexpressed in transgenic mice. Since then, the isolation and characterization of the protein named osteoprotegerin (OPG) has stimulated much work in the fields of endocrinology, rheumatology, and immunology. OPG functions as a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL, or OPG ligand) and shares homologies with other members of the tumor necrosis factor receptor superfamily. OPG acts by competing with the receptor activator of nuclear factor-kappaB, which is expressed on osteoclasts and dendritic cells for specifically binding to RANKL. RANKL is crucially involved in osteoclast functions and bone remodeling as well as immune cell cross-talks, dendritic cell survival, and lymph node organogenesis. More recently, emerging evidence from in vitro studies and mouse genetics attributed OPG an important role in vascular biology. In fact, OPG could represent the long sought-after molecular link between arterial calcification and bone resorption, which underlies the clinical coincidence of vascular disease and osteoporosis, which are most prevalent in postmenopausal women and elderly people.

Circulating monocytes and T lymphocytes extravasate through the endothelium at sites of developing atheromatous lesions, where they tend to accumulate and mediate the progression of the disease. We have previously demonstrated the presence of an enzymatically degraded, nonoxidized form of LDL (E-LDL) in early human fatty streaks, which possesses major biological properties of an atherogenic lipoprotein. The effects of E-LDL on human endothelial cells have now been studied with respect to adhesion and transmigration of monocytes and T lymphocytes. E-LDL induced a rapid and dose-dependent selective adhesion of monocytes and T lymphocytes to endothelial cell monolayers within 30 minutes of incubation. Maximal increases in the number of adherent monocytes (8-fold) and of adherent T lymphocytes (4-fold) were observed after treatment with 50 microg/mL E-LDL. E-LDL was more active than oxidized LDL (ox-LDL), whereas native LDL produced only minor adhesive effects. Both E-LDL and ox-LDL enhanced transmigration of monocytes and of T lymphocytes through endothelial monolayers. Again, E-LDL was more potent than ox-LDL, inducing transmigration to a similar extent as N-formyl-Met-Leu-Phe. In endothelial cells, E-LDL stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cells adhesion molecule-1 (PECAM-1), P-selectin, and E-selectin with distinct kinetics. Analyses with blocking antibodies indicated that ICAM-1 and P-selectin together mediated approximately 70% of cell adhesion, whereas blocking of PECAM-1 had no effect on adhesion but reduced transmigration to less than 50% of controls. E-LDL also upregulated expression of ICAM-1 in human aortic smooth muscle cells, and this correlated with increased adhesion of T lymphocytes. E-LDL is thus able to promote the selective adhesion of monocytes and T lymphocytes to the endothelium, stimulate transmigration of these cells, and foster their retention in the vessel wall by increasing their adherence to smooth muscle cells. These findings underline the potential significance of E-LDL in the pathogenesis of atherosclerosis.

The urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c-fos/c-myc mRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor-independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.