Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles.

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy

The Relaxin Gene Knockout Mouse: A Model of Progressive Scleroderma

Relaxin is a peptide hormone with anti-fibrotic properties. To investigate the long-term effects of relaxin deficiency on the ageing skin, we compared structural changes in the skin of ageing relaxin-deficient (RLX-/-) and normal (RLX+/+) mice, by biochemical, histological, and magnetic resonance imaging analyses. Skin biopsies from RLX+/+ and RLX-/- mice were obtained at different ages and analyzed for changes in collagen expression and distribution. We demonstrated an age-related progression of dermal fibrosis and thickening in male and female RLX-/- mice, associated with marked increases in types I and III collagen. The increased collagen was observed primarily in the dermis of RLX-/- mice by 1 mo of age, and eventually superseded the hypodermal layer. Additionally, fibroblasts from the dermis of RLX-/- mice were shown to produce increased collagen in vitro. Recombinant human gene-2 (H2) relaxin treatment of RLX-/- mice resulted in the complete reversal of dermal fibrosis, when applied to the early onset of disease, but was ineffective when applied to more established stages of dermal scarring. These combined findings demonstrate that relaxin provides a means to regulate excessive collagen deposition in disease states characterized by dermal fibrosis and with our previously published work demonstrate the relaxin-null mouse as a model of progressive scleroderma

Relaxin-1–deficient mice develop an age-related progression of renal fibrosis

Relaxin-1–deficient mice develop an age-related progression of renal fibrosis.BackgroundRelaxin (RLX) is a peptide hormone that stimulates the breakdown of collagen in preparation for parturition and when administered to various models of induced fibrosis. However, its significance in the aging kidney is yet to be established. In this study, we compared structural and functional changes in the kidney of aging relaxin-1 (RLX-/-) deficient mice and normal (RLX+/+) mice.MethodsThe kidney cortex and medulla of male and female RLX+/+ and RLX-/- mice at various ages were analyzed for collagen content, concentration, and types. Histologic analysis, reverse transcription-polymerase chain reaction (RT-PCR) of relaxin and relaxin receptor mRNA expression, receptor autoradiography, glomerular isolation/analysis, and serum/urine analysis were also employed. Relaxin treatment of RLX-/- mice was used to confirm the antifibrotic effects of the peptide.ResultsWe demonstrate an age-related progression of renal fibrosis in male, but not female, RLX-/- mice with significantly (P < 0.05) increased tissue dry weight, collagen (type I) content and concentration. The increased collagen expression in the kidney was associated with increased glomerular matrix and to a lesser extent, interstitial fibrosis in RLX-/- mice, which also had significantly increased serum creatinine (P < 0.05) and urinary protein (P < 0.05). Treatment of RLX-/- mice with relaxin in established stages of renal fibrosis resulted in the reversal of collagen deposition.ConclusionThis study supports the concept that relaxin may provide a means to regulate excessive collagen deposition during kidney development and in diseased states characterized by renal fibrosis

Lobe-specific modulation of B16MET melanoma lung metastases by nephrilin peptide

Aim: Nephrilin peptide modulates systemic immune responses to trauma in contexts characterized by simultaneous inflammation and immunosuppression. This study explores the possibility that nephrilin peptide may modulate lung metastasis, which also occurs in an environment of concurrent inflammation and immunosuppression.Methods: B16MET melanoma cells were injected via the tail vein of mice and the development of lung metastases was recorded in animals treated with nephrilin peptide or vehicle by subcutaneous bolus injection daily for three weeks. In a separate experiment, nephrilin was administered by subcutaneous bolus injection for seven days to study the biodistribution of peptide and possible changes to plasma cytokine levels.Results: Nephrilin significantly suppressed B16MET lung metastases. Suppression was more effective in deep lobes with the poorest access to circulation: accessory &gt; inferior &gt; middle &gt; superior. In a separate biodistribution study in mice, nephrilin showed similar biodistribution levels in kidney, liver, brain, and left lung, but significantly higher accumulation in the lobes of the right lung in a gradient that matched its effectiveness in suppressing metastases (accessory &gt; inferior &gt; middle). The latter environments were also characterized by significantly higher local concentrations of succinate, a proxy for lower levels of oxygenation.Conclusion: Nephrilin accumulates preferentially in the deep lobes of the right lung in mice and inhibits B16MET right lung metastases in a lobe-specific manner

Cytokines and Growth Factors Positively and Negatively Regulate Interstitial Collagen Gene Expression in Human Vascular Smooth Muscle Cells

Human atheromas accumulate extracellular matrix proteins such as collagen types I and III. We tested whether cytokines or growth factors produced by cells found in human atherosclerotic plaques alter collagen gene expression in vascular smooth muscle cells (VSMCs), which produce the blood vessel matrix. Interleukin-1 (IL-1, 1-10 ng/ml) modestly increased the synthesis of collagens I and III (measured by tritiated proline incorporation into specific electrophoretic bands), whereas transforming growth factor-$\beta$ (TGF-$\beta$) or platelet-derived growth factor (PDGF) markedly stimulated production of these interstitial collagens. Interferon $\gamma$ (IFN-$\gamma$), a product of activated T cells found in atheromas, selectively alters several VSMC functions. For example, this cytokine reduces growth of VSMCs, decreases alpha-actin gene expression, and induces VSMC expression of class II histocompatibility antigens. We report here that IFN-$\gamma$ also inhibits basal as well as IL-1-, PDGF-, or TGF-$\beta$-stimulated collagen I and III synthesis by human VSMCs. TGF-$\beta$, the most potent stimulator of collagen synthesis studied here, raised the level of collagen III mRNA in VSMCs 4.8-fold (determined by densitometry of Northern blots), whereas exposure to both TGF-$\beta$ and IFN-$\gamma$ reduced this mRNA to 0.5 of basal level. Locally produced cytokines and growth factors may thus modify matrix accumulation during atherogenesis by stimulating or suppressing expression of interstitial collagen mRNA and protein by VSMCs

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles.

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy