Valery N. Bochkov

Expression of cell adhesion molecule T-cadherin in the human vasculature.

Abstract. Alterations in expression of surface adhesion molecules on resident vascular and blood-derived cells play a fundamental role in the pathogenesis of cardiovascular disease. Smooth muscle cells (SMCs) have been shown to express T-cadherin (T-cad), an unusual GPI-anchored member of the cadherin family of adhesion molecules. Particular relevance for T-cad in cardiovascular tissues is indicated by our present screen (immunoblotting) of human tissues and organs whereby highest expression of T-cad was found in aorta, carotid, iliac and renal arteries and heart. To explore the (patho)physiological role for T-cad in the vasculature we performed an immunohistochemical analysis of T-cad expression in normal human aorta and atherosclerotic lesions of varying severity. T-cad was present both in the intima and media and was expressed in endothelial cells (ECs), SMCs and pericytes, but not in monocytes/macrophages, foam cells and lymphocytes. In the adventitia T-cad was present in the wall of vasa vasorum and was expressed in ECs, SMCs and pericytes. T-cad was differentially expressed in SMCs from distinct vascular layers of normal aorta (for example, high in the subendothelial (proteoglycan) layer of the intima, low in the musculoelastic intimal layer and in the media), as well as at different stages of lesion progression. In SMCs there was an apparent inverse relationship between the intensities of T-cad and smooth muscle α-actin expression, this being most prominent in lesions. The findings suggest a phenotype-associated expression of T-cad which may be relevant to control of the normal vascular architecture and its remodelling during atherogenesis.

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis

Abstract. Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1–4xa0days) after injury, and later (dayxa07–28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.

Density- and proliferation status-dependent expression of T-cadherin, a novel lipoprotein-binding glycoprotein: a function in negative regulation of smooth muscle cell growth?

The atypical low density lipoprotein (LDL) binding proteins (M r 105 and 130 kDa; p105 and p130) in human aortic medial membranes and cultured human and rat aortic smooth muscle cells (SMC) have recently been identified as the cell adhesion glycoprotein T‐cadherin. Although cadherins are generally recognized to be important regulators of morphogenesis, the function of T‐cadherin in the vasculature is poorly understood. This study has examined the relationship between expression of T‐cadherin and the density and proliferation status of SMC. T‐cadherin (p105 and p130) levels in SMC lysates were measured on Western blots using ligand‐binding techniques. T‐cadherin expression was dependent upon cell density, and maximal levels were achieved at confluency. T‐cadherin levels were reversibly modulated by switching cultures between serum‐free (upmodulation) and serum‐containing (downmodulation) conditions. Platelet‐derived growth factor (PDGF)‐BB, epidermal growth factor (EGF) or insulin‐like growth factor (IGF) elicited a dose‐ and time‐dependent downmodulation that was reversible after transfer of SMC to growth factor‐free medium. Our results support the hypothesis that T‐cadherin may function as a negative determinant of cell growth.

LDL binds to surface-expressed human T-cadherin in transfected HEK293 cells and influences homophilic adhesive interactions.

T‐cadherin (T‐cad) is an unusual glycosylphosphatidylinositol‐anchored member of the cadherin family of cell adhesion molecules. Binding of low density lipoproteins (LDLs) to T‐cad can be demonstrated on Western blots of smooth muscle cell lysates, membranes and purified proteins. Using HEK293 cells transfected with human T‐cad cDNA (T‐cad+), we have investigated the adhesion properties of expressed mature and precursor proteins and examined the postulate that LDL represents a physiologically relevant ligand for T‐cad. T‐cad+ exhibits an increased Ca2+‐dependent aggregation (vs. control) that was reduced by selective proteolytic cleavage of precursor T‐cad and abolished after either proteolytic or phosphatidylinositol‐specific phospholipase C (PI‐PLC) cleavage of both mature and precursor proteins, indicating that both proteins function in intercellular adhesion. T‐cad+ exhibited a significantly increased specific cell surface‐binding of [125I]‐LDL that was sensitive to PI‐PLC pre‐treatment of cells. Ca2+‐dependent intercellular adhesion of T‐cad+ was significantly inhibited by LDL. Our results support the suggestion that LDL is a physiologically relevant ligand for T‐cad.

LOW- AND HIGH-DENSITY-LIPOPROTEINS AS MITOGENIC FACTORS FOR VASCULAR SMOOTH-MUSCLE CELLS - INDIVIDUAL, ADDITIVE AND SYNERGISTIC EFFECTS

The mitogenic activities of low (LDL)- and high (HDL)-density lipoproteins have been examined in cultures of human vascular smooth muscle cells (VSMC). LDL and HDL3 dose-dependently (EC50 values approximately 50 micrograms/ml) stimulated DNA and protein synthesis ([3H]-thymidine and [3H]-leucine incorporation, respectively) in the absence of exogenously added mitogens. The synthetic responses of VSMC to combinations of LDL and HDL3 were additive, indicating that each lipoprotein mediates discrete effects. LDL or HDL3 promoted VSMC proliferation under strict mitogen-free conditions, but this growth response was not sustained. VSMC exposed to combinations of lipoproteins (either LDL or HDL3) and growth factors (either PDGF-BB, EGF, bFGF or IGF) exhibited synergistic DNA synthesis responses. In the combined presence of PDGF-BB and either LDL or HDL3, VSMC proliferation was sustained. Anionized lipoprotein preparations (oxidized, acetylated, carbamylated or malonimylated) also stimulated DNA and protein synthesis. Since the antioxidant beta-hydroxylated toluene did not block the effect of native LDL on DNA synthesis, and fucoidin, a specific competitor for the scavenger receptor, did not inhibit oxidized LDL-induced DNA synthesis, activation of mitogenic signals by lipoproteins does not depend on lipid peroxidation. Rather, the apparent intrinsic mitogenic potential of lipoproteins may depend upon their direct activation of replication-coupled signal transduction systems.

Identification of 130 kDa cell surface LDL-binding protein from smooth muscle cells as a partially processed T-cadherin precursor.

Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.

Platelets in patients with homozygous familial hypercholesterolemia are sensitive to Ca2+-mobilizing activity of low density lipoproteins

While some data suggest that Ca(2+)-mobilizing effects of low density lipoprotein (LDL) in platelets are mediated by a specific membrane receptor, the data about the nature of this lipoprotein-binding site are contradictory. This work was performed in order to assess possible involvement of apolipoprotein (apo) B,E receptor, present in most cell types. To answer the question we compared effects of LDL in normal platelets and those obtained from patients with homozygous familial hypercholesterolemia (HFH), characterized by absence of functional apo B,E receptors. We have found that in accordance with previous results LDL induced instant reversible elevation of free cytoplasmic calcium concentration ([Ca2+]i) in fura-2-loaded platelets. The effect was observed both in healthy and HFH groups. Neither half-maximal effective concentrations nor maximal effects of LDL differed significantly between two groups. Ca(2+)-mobilizing effects of lipoproteins were potentiated about 4-fold by epinephrine and completely blocked by prostaglandin E1 both in platelets of healthy and HFH subjects. The similarity of lipoprotein effects in control and HFH platelets is evidence that apo B,E receptor does not mediate the Ca(2+)-mobilizing activity of LDL in this cell type.

LDL- and agonist-induced Ca2+-mobilization in platelets of healthy subjects and in patients with familial hyperlipoproteinemia type II

LDL induced a reversible increase in [Ca2+]i in platelets of healthy subjects and FH-patients. In both groups the effects of LDL were potentiated 2.3-fold by epinephrine. [Ca2+]i increases, induced by LDL, ADP and PAF were more prominent in platelets of FH-patients. This may explain the platelet hyperaggregability in such conditions.

Epinephrine potentiates activation of human platelets by low density lipoproteins

Low density lipoproteins activate phosphoinositide turnover, increase free cytoplasmic calcium concentration and stimulate phosphorylation of 20- and 47-kDa proteins in blood platelets. All these effects are substantially potentiated by epinephrine.