Chrishan S. Samuel

Reduced nephron number in adult sheep, hypertensive as a result of prenatal glucocorticoid treatment.

There is some evidence, mainly from rodent studies, that any factor which alters the final total number of nephrons formed, during nephrogenesis, will result in hypertension in adult life. Sheep, programmed to become hypertensive by exposure to synthetic glucocorticoid (dexamethasone, 0.48 mg h−1, for 48 h) early in development (∼27 days of gestation), were killed at 7 years of age, and had nephron counting performed by unbiased stereology. Mean arterial pressure was 83 ± 4 mmHg in the dexamethasone (DEX) group (n= 5), and 73 ± 5 in the control (CON; n= 7; P < 0.05). The total nephron number, in the right kidney (249 070 ± 14 331; n= 5) was significantly lower (P < 0.01) than that of controls (402 787 ± 30 458; n= 7). Mean glomerular volume was larger in the DEX than the CON group (P < 0.01), but there was no significant difference in the sclerosis index between the two groups. Low nephron number was associated with grossly enlarged and dilated proximal tubules and greater accumulation of collagen type I and type III in the tubular interstitium and periadventitia of the renal cortical vessels. These data suggest that the hypertensive programming effect of glucocorticoid treatment, early in kidney development, results, at least in part, from impaired nephrogenesis.

Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin-induced lung injury.

Acute respiratory distress syndrome is characterized by loss of lung tissue as a result of inflammation and fibrosis. Augmenting tissue repair by the use of mesenchymal stem cells may be an important advance in treating this condition. We evaluated the role of term human umbilical cord cells derived from Whartons jelly with a phenotype consistent with mesenchymal stem cells (uMSCs) in the treatment of a bleomycin-induced mouse model of lung injury. uMSCs were administered systemically, and lungs were harvested at 7, 14, and 28 days post-bleomycin. Injected uMSCs were located in the lung 2 weeks later only in areas of inflammation and fibrosis but not in healthy lung tissue. The administration of uMSCs reduced inflammation and inhibited the expression of transforming growth factor-beta, interferon-gamma, and the proinflammatory cytokines macrophage migratory inhibitory factor and tumor necrosis factor-alpha. Collagen concentration in the lung was significantly reduced by uMSC treatment, which may have been a consequence of the simultaneous reduction in Smad2 phosphorylation (transforming growth factor-beta activity). uMSCs also increased matrix metalloproteinase-2 levels and reduced their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases, favoring a pro-degradative milieu following collagen deposition. Notably, injected human lung fibroblasts did not influence either collagen or matrix metalloproteinase levels in the lung. The results of this study suggest that uMSCs have antifibrotic properties and may augment lung repair if used to treat acute respiratory distress syndrome.

Relaxin Reverses Cardiac and Renal Fibrosis in Spontaneously Hypertensive Rats

The antifibrotic effects of the peptide hormone relaxin on cardiac and renal fibrosis were studied in 9- to 10-month-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Rats (n=8 to 9 per group) were allocated into 3 groups: WKY controls, vehicle-treated SHR (SHR-V), and relaxin-treated SHR (SHR-R). Relaxin (0.5 mg/kg per day) was administered via subcutaneously implanted osmotic mini-pumps over 2 weeks before hearts and kidneys were harvested for analysis. Collagen content was analyzed by hydroxyproline assay, gel electrophoresis, and quantitative histology. Zymography was used to determine matrix metalloproteinase (MMP) expression and Western blotting to determine proliferating cell nuclear antigen (PCNA) expression and α-smooth muscle actin (α-SMA)/myofibroblast expression, whereas cardiac hypertrophy was assessed by myocyte size and real-time polymerase chain reaction of associated genes. The left ventricular (LV) myocardium of SHR-V contained increased collagen levels (by 25±1%, P<0.01 using biochemical analysis and 3-fold; P<0.01 using quantitative histology), enhanced expression of PCNA (by 70±8%; P<0.01), α-SMA (by 32±2%; P<0.05), and the collagen-degrading enzyme MMP-9 (by 70±6%; P<0.05) versus respective levels measured in WKY controls. The kidneys of SHR-V also contained increased collagen (25±2%, P<0.05 using biochemical analysis and 2.4-fold; P<0.01 using quantitative histology). Relaxin treatment significantly normalized collagen content in the LV (P<0.01) and kidney (P<0.05), completely inhibited cell proliferation (P<0.01) and fibroblast differentiation (P<0.05) in the LV, and increased MMP-2 expression (by 25±1%; P<0.05) without affecting MMP-9 in the LV compared with that measured in SHR-V. Thus, relaxin is a potent antifibrotic hormone with a rapid-occurring efficacy that may have therapeutic potential for hypertensive disease.

Relaxin deficiency in mice is associated with an age-related progression of pulmonary fibrosis

Relaxin (RLX) is a peptide hormone with known antifibrotic properties. However, its significance in the lung and its role as a therapeutic agent against diseases characterized by pulmonary fibrosis are yet to be established. In this study, we examined age‐related structural and functional changes in the lung of relaxin‐deficient mice. Lung tissues of male and female RLX knockout (–/–) and RLX wild‐type (+/+) mice at various ages were analyzed for changes in collagen expression and content. We demonstrate an age‐related progression of lung fibrosis in RLX –/– mice with significantly increased tissue wet weight, collagen content and concentration, alveolar congestion, and bronchiole epithelium thickening. The increased fibrosis was associated with significantly altered peak expiratory flow and lung recoil (lung function) in RLX –/– mice. Treatment of RLX –/– mice with relaxin in early and developed stages of fibrosis resulted in the reversal of collagen deposition. Organ bath studies showed that precontracted lung strips relaxed in the presence of relaxin. Together, these data indicate that relaxin may provide a means to regulate excessive collagen deposition in diseased states characterized by pulmonary fibrosis.

Obesity results in progressive atrial structural and electrical remodeling: implications for atrial fibrillation

BACKGROUND Obesity is associated with atrial fibrillation (AF); however, the mechanisms by which it induces AF are unknown. OBJECTIVE To examine the effect of progressive weight gain on the substrate for AF. METHODS Thirty sheep were studied at baseline, 4 months, and 8 months, following a high-calorie diet. Ten sheep were sampled at each time point for cardiac magnetic resonance imaging and hemodynamic studies. High-density multisite biatrial epicardial mapping was used to quantify effective refractory period, conduction velocity, and conduction heterogeneity index at 4 pacing cycle lengths and AF inducibility. Histology was performed for atrial fibrosis, inflammation, and intramyocardial lipidosis, and molecular analysis was performed for endothelin-A and -B receptors, endothelin-1 peptide, platelet-derived growth factor, transforming growth factor β1, and connective tissue growth factor. RESULTS Increasing weight was associated with increasing left atrial volume (P = .01), fibrosis (P = .02), inflammatory infiltrates (P = .01), and lipidosis (P = .02). While there was no change in the effective refractory period (P = .2), there was a decrease in conduction velocity (P<.001), increase in conduction heterogeneity index (P<.001), and increase in inducible (P = .001) and spontaneous (P = .001) AF. There was an increase in atrial cardiomyocyte endothelin-A and -B receptors (P = .001) and endothelin-1 (P = .03) with an increase in adiposity. In association, there was a significant increase in atrial interstitial and cytoplasmic transforming growth factor β1 (P = .02) and platelet-derived growth factor (P = .02) levels. CONCLUSIONS Obesity is associated with atrial electrostructural remodeling. With progressive obesity, there were changes in atrial size, conduction, histology, and expression of profibrotic mediators. These changes were associated with spontaneous and more persistent AF.

Increased myocardial collagen and ventricular diastolic dysfunction in relaxin deficient mice: a gender-specific phenotype

OBJECTIVE To investigate cardiac phenotypes in mice deficient in the peptide hormone relaxin by gene targeting. METHODS Echocardiography and cardiac catheterization were performed on male and female relaxin deficient (Rlx(-/-)) mice as well as heterozygous (Rlx(+/-)) and wildtype (Rlx(+/+)) littermates aged between 8 and 24 months. Collagen expression and content in the heart were analysed by real-time PCR, hydroxyproline assay and histology. RESULTS Heart rate, blood pressures, left ventricular (LV) dimensions, fractional shortening and maximal and minimal dP/dt did not differ significantly between the three genotypes of either gender at any age. However, 8-10-month-old Rlx(-/-) males exhibited a greater transmitral flow velocity (A-wave) at the late LV diastolic phase. Male Rlx(-/-) mice aged between 12 and 24 months had significantly higher LV end-diastolic pressures, a 30% increase in atrial weight and 10-30% increases in lung and liver weights. Male mice also showed an age-dependent increase (P<0.01) in LV collagen content that was more pronounced in Rlx(-/-) than control littermates (P<0.01). Procollagen type-1 expression was also significantly higher in the LV of Rlx(-/-) males compared with either Rlx(+/-) or Rlx(+/+) males at 6, 9 and 12 months of age. Age-matched female Rlx(-/-) mice did not display any of these cardiac phenotypes seen in Rlx(-/-) males. CONCLUSIONS Male Rlx(-/-) mice had impeded LV diastolic filling and increased atrial weights, most likely due to an increase in ventricular collagen content and chamber stiffness. These phenotypes in the Rlx(-/-) males were not observed in Rlx(-/-) females, indicating the importance of other gender-related factors in cardiovascular function.

Renal Structural and Functional Repair in a Mouse Model of Reversal of Ureteral Obstruction

The end point of immune and nonimmune renal injury typically involves glomerular and tubulointerstitial fibrosis. Although numerous studies have focused on the events that lead to renal fibrosis, less is known about the mechanisms that promote cellular repair and tissue remodeling. Described is a model of renal injury and repair after the reversal of unilateral ureteral obstruction (UUO) in male C57bl/6J mice. Male mice (20 to 25 g) underwent 10 d of UUO with or without 1, 2, 4, or 6 wk of reversal of UUO (R-UUO). UUO resulted in cortical tubular cell atrophy and tubular dilation in conjunction with an almost complete ablation of the outer medulla. This was associated with interstitial macrophage infiltration; increased hydroxyproline content; and upregulated type I, III, IV, and V collagen expression. The volume density of kidney occupied by renal tubules that exhibited a brush border was measured as an assessment of the degree of repair after R-UUO. After 6 wk of R-UUO, there was an increase in the area of kidney occupied by repaired tubules (83.7 +/- 5.9%), compared with 10 d UUO kidneys (32.6 +/- 7.3%). This coincided with reduced macrophage numbers, decreased hydroxyproline content, and reduced collagen accumulation and interstitial matrix expansion, compared with obstructed kidneys from UUO mice. GFR in the 6-wk R-UUO kidneys was restored to 43 to 88% of the GFR in the contralateral unobstructed kidneys. This study describes the regenerative potential of the kidney after the established interstitial matrix expansion and medullary ablation associated with UUO in the adult mouse.

Collagen Studies in Late Pregnant Relaxin Null Mice

Abstract The relaxin knockout (rlx −/−) mouse was used to assess the effect, during pregnancy, of relaxin with regard to water, collagen content, growth, and morphology of the nipple (N), vagina (V), uterus, cervix (C), pubic symphysis (PS), and mammary gland (MG). The results presented here indicate that during pregnancy, relaxin increases the growth of the N, C, V, and PS. Large increases in water content in the PS (20%) occurred in pregnant (Day 18.5) wild-type (rlx +/+) mice but not in rlx −/− animals. This indicates that in the PS, relaxin might increase the concentration of a water-retaining extracellular matrix component (hyaluronate). In the pregnant rlx +/+ mouse, collagen content decreased significantly in the N and V but not in other tissues. There were no significant changes in the rlx −/− mouse. This contrasts with findings in the rat, in which relaxin has been found to cause decreases in collagen concentrations in the V, C, and PS. Histological analysis showed that the collagen stain was more condensed in the tissues (V, C, PS, N, and MG) of rlx −/− mice than in those of rlx +/+ mice. This phenomenon indicates that the failure of collagen degradation and lack of growth in the N underlie the inability of the rlx −/− mice to feed their young, as reported previously. Vaginal and cervical luminal epithelia, which proliferated markedly in the rlx +/+ pregnant mice, remained relatively atrophic in the rlx −/− mice. As proliferation and differentiation of uterine and vaginal epithelia are thought to be induced by a paracrine stromal factor that acts upon estrogen stimulation, our results indicate that relaxin may be this paracrine factor.

Cardiovascular effects of relaxin: from basic science to clinical therapy.

Although substantial advances have been achieved in recent decades in the clinical management of heart diseases, new therapies that provide better or additional efficacy with minimal adverse effects are urgently required. Evidence that has accumulated since the 1990s indicates that the peptide hormone relaxin has multiple beneficial actions in the cardiovascular system under pathological conditions and, therefore, holds promise as a novel therapeutic intervention. Clinical trials for heart failure therapy using relaxin revealed several beneficial actions. Here we review findings from mechanistic and applied research in this field, comment on the outcomes of recent phase I/II clinical trails on patients with heart failure, and highlight settings of cardiovascular diseases where relaxin might be effective.

Transplantation of Human Amnion Epithelial Cells Reduces Hepatic Fibrosis in Immunocompetent CCl4-Treated Mice:

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.