Clusterin (16. Vasculature)

Smooth Muscle

Cultured porcine smooth muscle cells (SMC) undergo morphological and phenotypic modulation associated with a change from a substrate-attached monolayer culture to a nodular culture in which most of the cells are present in multicellular aggregations (nodules). During that transition from monolayer to nodular cell culture (> 8 days) the expression of clusterin mRNA and protein is increased. Clusterin expression continues in the nodular cell cultures and it is secreted at 0.3 microgram­s/ml/24 h. These results demonstrate differential expression of SMC clusterin and suggest that clusterin has a functional role in SMC modulation ^Ref .

Clusterin, at 10 microg/ml, clearly promotes vascular smooth Musile cells (VSMC) migration. In addition, a 15 amino acid synthetic peptide, representing amino acids 118–132 [KQTCMKFYAEVCRSG] of the mature clusterin polypeptide, inhibits VSMC attachment to gelatinous substrate. Finally, clusterin appears to have a role in regulating endogenous clusterin expression in the clusterin negative clone. These results clearly establish that clusterin has functional role in VSMC nodule formation and support the conclusion that clusterin is a critical component of smooth muscle cell phenotypic modulation ^Ref .
Clusterin mRNA was not detectable in noninjured aorta (control), began to be expressed at 6 hours after injury, showed a peak level at 24 hours (a 48-fold increase), gradually declined, and returned to the control level at 24 weeks.Western blot and immunohistoche­mistry demonstrated no expression of clusterin protein in noninjured aorta, an expression of clusterin at 2 days after balloon injury,and a peak level (a 55-fold increase) at 2 to 8 weeks.the expression of clusterin protein continued until 24 weeks after injury. In situ hybridization revealed that clusterin mRNA was expressed in smooth muscle cells (SMCs) of media at 2 days after injury and in SMCs of media and neointima at 2 weeks.to analyze the function of clusterin,stably transfected rabbit SMCs were created. The expression of clusterin stimulated proliferation and migration of SMCs. Clusterin is dramatically induced in media and neointima after vascular injury, suggesting that clusterin contributes to restenosis after angioplasty ^Ref .

Intimal Cells

Clusterin expression was evaluated by immunohistoche­mistry and Western blotting in human arteries and rat aortas. In human diffuse myointimal thickening, clusterin was detected in cell cytoplasm and extracellular space, whereas it was practically absent in the media. In rat aortas 15 days after ballooning, intimal cells (IT cells) overexpressed s-clusterin and n-clusterin, the latter mainly in the inner neointima; clusterin expression decreased at 60 days. In vitro, IT cells maintained high clusterin expression and its antisense markedly reduced proliferation and increased apoptosis. Western blotting showed that all-trans retinoic acid-induced proliferative arrest and increased alpha-smooth muscle actin expression did associate to s-clusterin and B-myb reduction, whereas bax-related apoptosis was associated to a shift from the s-clusterin to n-clusterin isoform. Clusterin overexpression characterized neointimal SMCs; s-clusterin expression decreased in IT cells during all-trans retinoic acid-induced proliferative arrest and redifferentiation, whereas n-clusterin overexpression was characteristic of apoptosis. Clusterin was detected in human arterial myointimal thickening and absent in the underlying media. Rat neointimal cells overexpressed clusterin and clusterin antisense oligonucleotide reduced proliferation and increased apoptosis.Alltrans retinoic acid-induced proliferative arrest showed association with sclusterin reduction and n-clusterin overexpression with apoptosis, supporting a different biological role of these isoforms ^Ref .

Endothelial cells

Clusterin inhibits HUVEC migration and adhesion. By altering endothelial function during vascular injury, clusterin appears to regulate, in part, the early development of intimal hyperplasia after prosthetic arterial grafting ^Ref .

When Human retinal endothelial cells (HRCs) were exposed to oxygen-glucose deprivation (OGD), clusterin expression increased, whereas von Willebrand factor (vWF), occludin, and zonula occludens (ZO-1) markedly decreased. Interestingly, loss of tight junction proteins and death of HRCs in OGD conditions were restored by clusterin treatment. These results suggest that the enhanced clusterin in OGD conditions may play a protective role against ischemia-induced tight junction protein loss and HRCs death ^Ref .

GIT

As compared with controls, a strongly enhanced expression of clusterin was found in Crohn disease (CD) tissues, correlating with disease activity. Immunohistoche­mistry and in situ hybridization analysis revealed foci of crypts almost completely lined by clusterin expressing enterocytes in CD, a feature that was never seen in controls. Such crypts appeared especially within the morphologically intact mucosa apart from erosive or ulcerative lesions. Besides epithelia, clusterin was also expressed by inflammatory mononuclear cells. Enhanced expression of clusterin by crypt epithelia might reflect a cytoprotective function of the protein in order to prevent further injury of the intestinal mucosal barrier in CD ^Ref .

Connective Tissue

The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced. The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition ^Ref .

in synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in reumatoid arthritis (RA) tissues compared with osteoarthritis (OA) and healthy synovium. In synovium and in cultured FLS, the overexpression of clusterin concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of clusterin in RA FLS promoted apoptosis within 24 h. It was observed that clusterin knockdown with small interfering RNA promoted IL-6 and IL-8 production. Clusterin interacted with phosphorylated ikappaBalpha. Differential expression of clusterin by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. It was proposed that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes ^Ref .

Fibroblasts

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest,an increase in the senescence-associated beta-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as clusterin. Clusterin has been described as a survival gene against cytotoxic stress. In order to study whether clusterin would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of clusterin was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of clusterin resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing clusterin. No effect of clusterin overexpression was observed on the proliferative life span of HDFs, even if clusterin overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Clusterin sene-scence-related overexpression is proposed to have antiapoptotic rather than antiproliferative effects ^Ref .

Fibroblasts were coincubated with conditioned medium and cigarette smoke extract, and bronchial biopsy specimens obtained from nonsmokers, smokers, and ex-smokers were analyzed by immunohistoche­mistry. At concentrations of 2.5 and 5.0%, cigarette smoke extract induced oxidative stress. It also markedly increased the expression of two clusterin isoforms (60 and 7680 kD) and the 76–80-kD isoform was secreted in the incubation medium. Coincubation of fibroblasts with conditioned medium significantly decreased the cellular oxidation caused by the cigarette smoke extract. Immunohistochemical analysis of clusterin on bronchial biopsy specimens obtained from smokers and ex-smokers showed localization of clusterin mainly in the submucosa. I was concluded that clusterin may have a protective effect against cigarette smoke-induced oxidative stress in lung fibroblasts ^Ref .

---