Marta Otero-Viñas

Low-Density Lipoprotein Upregulates Low-Density Lipoprotein Receptor-Related Protein Expression in Vascular Smooth Muscle Cells. Possible Involvement of Sterol Regulatory Element Binding Protein-2-Dependent Mechanism

Background—Low-density lipoprotein (LDL) receptor-related protein (LRP) is highly expressed in vascular smooth muscle cells (VSMCs) of both normal and atherosclerotic lesions. However, little is known about LRP regulation in the vascular wall. Methods and Results—We analyzed the regulation of LRP expression in vitro in human VSMCs cultured with native LDL (nLDL) or aggregated LDL (agLDL) by semiquantitative reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, and Western blot and in vivo during diet-induced hypercholesterolemia by in situ hybridization. LRP expression in human VSMCs is increased by nLDL and agLDL in a time- and dose-dependent manner. Maximal induction of LRP mRNA expression was observed after 24 hours of exposure to LDL. However, agLDL induced higher LRP mRNA expression (3.0-fold) than nLDL (1.76-fold). LRP mRNA upregulation was associated with an increase on LRP protein expression with the greatest induction by agLDL. VSMC-LRP upregulation induced by nLDL or agLDL was reduced by an inhibitor of sterol regulatory element binding protein (SREBP) catabolism (N-acetyl-leucyl-leucyl-norleucinal). In situ hybridization analysis indicates that there is a higher VSMC-LRP expression in hypercholesterolemic than in normocholesterolemic pig aortas. Conclusions—These results indicate that LRP expression in VSMCs is upregulated by intravascular and systemic LDL.

Cholesteryl Esters of Aggregated LDL Are Internalized by Selective Uptake in Human Vascular Smooth Muscle Cells

Objective—Low-density lipoprotein (LDL) receptor-related protein (LRP1) mediates the internalization of aggregated LDL (agLDL)–LDL trapped in the arterial intima bound to proteoglycans–into human vascular smooth muscle cells (VSMC). LRP1-mediated agLDL uptake induces high-intracellular cholesteryl ester (CE) accumulation. The aim of this study was to characterize the mechanism of agLDL internalization in human VSMC. Methods and Results—The lipidic component of LDL was labeled with [3H] and the apolipoprotein component with [125I]. We found that >90% of intracellular CE derived from agLDL uptake was not associated with apoB100 degradation but was selectively taken up from agLDL. The inhibition of LRP1 expression by small interfering RNA treatment led to a decrease of 80±0.05% in agLDL-CE selective uptake. AgLDL induced intracellular CE accumulation without a concomitant CE synthesis. Cytosolic and cytoskeletal proteins were not required for CE transport. Electron and confocal microscopy experiments indicate that CE derived from agLDL accumulated in adipophilin-stained lipid droplets that were not removable by high-density lipoprotein. Conclusions—Taken together, these results demonstrate that LRP1 mediates the selective uptake of CE from agLDL and that CE derived from agLDL is not intracellularly processed but stored in lipid droplets in human VSMC.

Differential Role of Heparan Sulfate Proteoglycans on Aggregated LDL Uptake in Human Vascular Smooth Muscle Cells and Mouse Embryonic Fibroblasts

Objective—Low density lipoprotein (LDL) receptor–related protein (LRP) binds and internalizes aggregated LDL (agLDL) in human vascular smooth muscle cells (VSMCs). To analyze the contribution of proteoglycans (PGs) to agLDL uptake in human VSMCs, in wild-type mouse embryonic fibroblasts (MEF line), and in LRP-deficient mouse embryonic fibroblasts (PEA13 line). Methods and Results—PGs in the medium and cellular and extracellular matrix have been isolated by metabolic radiolabeling with [35S]Na2SO4 and characterized by selective digestion with heparinase I and III (4 U/mL each) and chondroitinase ABC (2 U/mL). To examine the contribution of PGs and LRPs to agLDL internalization, nonexpressing and LRP-expressing cells, treated or not with polysaccharidase, were incubated with agLDL (25, 50, and 100 &mgr;g/mL) for 18 hours. In human VSMCs, agLDL was unable to induce cholesteryl ester (CE) accumulation in antisense LRP-oligodeoxynucleotide–treated cells, and heparan sulfate (HS)-PG depletion leads to a reduction of the CE accumulation. In mouse fibroblasts, PEA13 compared with MEF showed lower, but still considerable, CE accumulation, and HS-PG depletion almost completely inhibited CE accumulation. Conclusions—In MEF, HS-PGs can function alone as receptors that bind and internalize agLDL in the absence of LRP, but in human VSMCs, although HS-PGs facilitate agLDL binding to the cells, LRP is essential for agLDL internalization.

Intracellular lipid accumulation, low-density lipoprotein receptor-related protein expression, and cell survival in vascular smooth muscle cells derived from normal and atherosclerotic human coronaries

Background  Vascular smooth muscle cell (VSMC) regulation during atherosclerotic plaque progression is determinant for plaque stability.

Transforming growth factor beta (TGF-β) isoforms in wound healing and fibrosis.

Scar formation, with persistent alteration of the normal tissue structure, is an undesirable and significant result of both wound healing and fibrosing disorders. There are few strategies to prevent or to treat scarring. The transforming growth factor beta (TGF‐β) superfamily is an important mediator of tissue repair. Each TGF‐β isoform may exert a different effect on wound healing, which may be context‐dependent. In particular, TGF‐β1 may mediate fibrosis in adults’ wounds, while TGF‐β3 may promote scarless healing in the fetus and reduced scarring in adults. Thus, TGF‐β3 may offer a scar‐reducing therapy for acute and chronic wounds and fibrosing disorders.

An in vitro priming step increases the expression of numerous epidermal growth and migration mediators in a tissue‐engineering construct

An FDA‐approved, prototypic, living, bilayered skin construct (BSC) has been used for non‐healing wounds. Using this particular construct as proof of principle, we hypothesized that an in vitro ‘priming’ step may enhance its repertoire of expression of key mediators and genes. The priming step used here was incubation in Dulbeccos modified Eagles medium (DMEM) for 24 h at 37°C and 5% CO2, with or without construct meshing. Microarray and ingenuity pathway analysis (IPA) showed that >1000 genes were overexpressed by the priming step, including interleukin 6 (IL‐6), which plays important roles in wound healing. Genes highly overexpressed by priming were those involved in epidermal proliferation and migration. Quantitative real‐time PCR (qRT–PCR), immunostaining and western blots verified the results. An epiboly assay (epidermal migration over dermis) showed that BSC epiboly was inhibited by IL‐6 neutralizing antibody. Back wounds of nude mice were treated with primed or control BSCs for 3 days prior to harvesting; primed BSCs showed a significantly (p = 0.006) greater level of epidermal migration vs unprimed. Our study demonstrates that an in vitro priming step induces wound healing‐related genes in the BSC, leading to a construct that could prove more effective in stimulating wound healing. Copyright

Liver Enzymes and Lipid Levels in Patients With Lipodermatosclerosis and Venous Ulcers Treated With a Prototypic Anabolic Steroid (Stanozolol) A Prospective, Randomized, Double-Blinded, Placebo-Controlled Trial

Anabolic steroids have been used to treat lower extremity ulcerations, including venous and cryofibrinogenemic ulcers and lipodermatosclerosis (LDS). Yet there have been no studies to determine the severity and reversibility of side effects of anabolic steroids on liver enzymes and lipid profiles in elderly patients. We therefore evaluated, in a prospective, randomized, double-blinded, placebo-controlled trial, the extent and reversibility of abnormal liver enzymes and lipid profiles in patients with LDS and venous leg ulcers treated with stanozolol at 2 mg twice daily for up to 6 months. Follow-up laboratory testing was done for 2 months after cessation of treatment. A total of 44 patients with LDS and venous ulcers were enrolled and treated with either leg compression alone (placebo) or leg compression plus oral stanozolol 2 mg twice daily (active). Baseline and follow-up laboratory testing of liver enzymes and lipid profiles were obtained. A total of 21 active and 23 placebo patients were treated and evaluated. We measured liver enzymes (aspartate aminotransferase [AST/SGOT], alanine aminotransferase [ALT/SGPT], γ-glutamyl transferase [GGT]) and lipid profile components (high-density lipoprotein [HDL], low-density lipoprotein [LDL], total cholesterol) before, during, and after the treatment period. We found that AST/SGOT and ALT/SGPT became significantly elevated in 29% (P = .0415 at 2 months) and 33% (P = .0182 at 1 month) of patients treated with stanozolol or placebo, respectively, with return to baseline in the posttreatment period. Unexpectedly, 91% of patients on stanozolol developed a significant (P < .0001) decrease in HDL levels, by as much as 37 U/L. All patients remained asymptomatic and levels returned to baseline after discontinuation of the drug. We conclude that low-dose stanozolol, 2 mg twice daily, produces asymptomatic and temporary elevation of liver transaminases and depression of the HDL level in a significant proportion of patients.