Tim D. Hewitson

Fibroblast Differentiation in Wound Healing and Fibrosis

The contraction of granulation tissue from skin wounds was first described in the 1960s. Later it was discovered that during tissue repair, fibroblasts undergo a change in phenotype from their normal relatively quiescent state in which they are involved in slow turnover of the extracellular matrix, to a proliferative and contractile phenotype termed myofibroblasts. These cells show some of the phenotypic characteristics of smooth muscle cells and have been shown to contract in vitro. In the 1990s, a number of researchers in different fields showed that myofibroblasts are present during tissue repair or response to injury in a variety of other tissues, including the liver, kidney, and lung. During normal repair processes, the myofibroblastic cells are lost as repair resolves to form a scar. This cell loss is via apoptosis. In pathological fibroses, myofibroblasts persist in the tissue and are responsible for fibrosis via increased matrix synthesis and for contraction of the tissue. In many cases this expansion of the extracellular matrix impedes normal function of the organ. For this reason much interest has centered on the derivation of myofibroblasts and the factors that influence their differentiation, proliferation, extracellular matrix synthesis, and survival. Further understanding of how fibroblast differentiation and myofibroblast phenotype is controlled may provide valuable insights into future therapies that can control fibrosis and scarring.

Renal tubulointerstitial fibrosis: common but never simple

Regardless of etiology, all patients with chronic renal disease show a progressive decline in renal function with time. Fibrosis, so-called scarring, is a key cause of this pathophysiology. Fibrosis involves an excess accumulation of extracellular matrix (primarily composed of collagen) and usually results in loss of function when normal tissue is replaced with scar tissue. While recent major advances have led to a much better understanding of this process, many problems remain. We for instance know little about why some wounds heal and others scar and little about how many putative antifibrotic agents work. This review discusses recent advances in our understanding of the mechanisms of tubulointerstitial fibrosis, focusing on the regulation and role of the myofibroblast in this process, the role of recently recognized endogenous antifibrotic factors, controversy surrounding the effects of metalloproteinases, and the opportunities presented by new treatment strategies that abrogate and may even reverse fibrosis.

Clinical and Histological Correlations of Decline in Renal Function in Diabetic Patients With Proteinuria

In 47 patients with diabetic nephropathy (29 type I, 18 type II) renal function and blood pressure (BP) (treated with or without an angiotensin-converting enzyme [ACE] inhibitor, enalapril [10 mg], in 38 hypertensive patients) were followed over 4 years. A percutaneous renal biopsy was performed in all patients initially and repeated in a representative 19 patients with treated hypertension after 4 years. Mean glomerular volume (MGV), interstitial fibrosis (IF), capillary volume, and sclerosed glomeruli (GS) were measured histomorphometrically. Mean fall in creatinine clearance (CCr) was 11.8% after 4 years with no difference between treatment groups or type of diabetes. BP both initially and during treatment correlated with initial and final serum creatinine and CCr (P < 0.01). There were no histomorphometric differences between type I and type II patients or hypertension treatment groups. Initial IF correlated with initial and final serum creatinine and CCr (P < 0.05) in all patients and type I patients alone, MGV correlated inversely with CCr in type I patients (P < 0.05). After 4 years, IF (24.8 vs. 30.0%, P < 0.01) and GS (26 vs. 37%, P < 0.05) increased significantly, and increase in IF correlated with fall in CCr (P < 0.01). Proteinuria and HbA1 did not correlate with indexes of function or structure. In this longitudinal study of patients with diabetic nephropathy, there was a close relation between BP and renal function but no difference between treatment with enalapril and other hypertensive agents. The correlations between renal function and histology at entry and after 4 years suggest that IF is a co-determinant of renal function in diabetic nephropathy.

The role of tubulointerstitial injury in chronic renal failure

Progressive renal failure results from a triad of glomerulosclerosis, tubulointerstitial fibrosis and vascular sclerosis. The mechanisms by which tubules are injured, and by which the tubular epithelial cell then excites interstitial inflammation culminating in fibroblast activation and fibrosis have become increasingly understood. Most current methods to prevent progressive glomerulosclerosis would inherently prevent tubular injury and interstitial fibrosis. The behaviour and control of the renal fibroblast is being investigated, with the potential for direct interference with its functions.

Parathyroid hormone has a prosclerotic effect on vascular smooth muscle cells

Although accelerated atherosclerosis and arteriosclerosis are common in patients with renal failure, the pathogenesis of these changes is poorly understood. Parathyroid hormone (PTH) levels are elevated in renal failure, and have been linked to uraemic vascular changes in some studies. We examined the in vitro effects of increasing doses of the 1–34 fragment of PTH on human aortic vascular smooth muscle cells (VSMCs). Factors examined were: (1) collagen production using tritiated hydroxyproline incorporation and transcription of procollagen α1(I) mRNA; (2) change in the surface area of collagen I lattices; (3) mRNA transcription of the collagen binding protein β1 integrin; (4) proliferation using tritiated thymidine incorporation, and (5) methyl tetrazolium salt conversion to estimate live cell number after 5 days’ exposure to PTH. PTH at a concentration of 200 pmol/l increased total collagen synthesis (188 ± 25% of control, p < 0.01) as well as transcription of procollagen α1(I) mRNA (136 ± 11% of control, p < 0.005). PTH also increased reorganisation of collagen I lattices (surface area 47 ± 8% of well for control vs. 35.7 ± 2.5 and 34.3 ± 3.0% for PTH 100 and 200 pmol/l, respectively, p = 0.02) and upregulated β1 integrin mRNA expression (160 ± 20% of control at PTH concentration of 200 pmol/l, p < 0.05). PTH had no effect on VSMC proliferation or number at doses up to 200 pmol/l. In conclusion, PTH increases production and reorganisation of collagen by VSMCs in vitro. It is possible that more aggressive control of hyperparathyroidism in patients with renal failure may help to reduce the burden of cardiovascular disease in this patient population.

Interstitial myofibroblasts in IgA glomerulonephritis.

We examined renal biopsy specimens from patients with mesangial IgA glomerulonephritis (n = 25; plasma creatinine 0.05-0.30 mmol/l) to ascertain whether the myofibroblast has a role in progressive renal interstitial fibrosis. Myofibroblasts were identified by morphology and alpha smooth muscle actin (alpha-SMA) immunostaining at the light and electron microscope level. Results were related to staining for interstitial leukocytes and collagen III. A control group consisted of 6 normal renal transplant donors from whom biopsy specimens were taken at the time of vascular anastomosis. The fractional volume of interstitial alpha-SMA staining was greater in patients with mesangial IgA glomerulonephritis than in the control group (17.2 vs. 1.3%; p < 0.001). alpha-SMA staining was increased in areas of interstitial fibrosis with prominent periglomerular and peritubular distribution. Ultrastructural studies established that alpha-SMA staining in the renal interstitium was intracellular, cytoplasmic, and confined to myofibroblast-like cells and processes. The alpha-SMA expression correlated with fractional volume of tubular atrophy/dilation (r = 0.79, p < 0.001), interstitial connective tissue (r = 0.66, p < 0.001), leucocytes (r = 0.72, p < 0.005), and collagen III (r = 0.71, p < 0.001). Staining correlated with renal function at the time of biopsy (r = 0.64, p < 0.005) and after 2 years of follow-up (r = 0.77, p < 0.01). In conclusion, cells with a myofibroblast-like phenotype have a significant role in the progression of tubulointerstitial injury.

Relaxin inhibits renal myofibroblast differentiation via RXFP1, the nitric oxide pathway, and Smad2

The hormone relaxin inhibits renal myofibroblast differentiation by interfering with TGF‐β1/ Smad2 signaling. However, the pathways involved in the relaxin‐TGF‐β1/Smad2 interaction remain unknown. This study investigated the signaling mechanisms by which human gene‐2 (H2) relaxin regulates myofibroblast differentiation in vitro by examining its effects on mixed populations of fibroblasts and myofibroblasts propagated from injured rat kidneys. Cultures containing ~60–70% myofibroblasts were used to determine which relaxin receptors, G‐proteins, and signaling pathways were involved in the H2 relaxin‐mediated regulation of α‐smooth muscle actin (α‐SMA; a marker of myofibroblast differentiation). H2 relaxin only inhibited α‐SMA immunostaining and collagen concentration in the presence of relaxin family peptide receptor 1 (RXFP1). H2 relaxin also induced a transient rise in cAMP in the presence of Gi/o inhibition, and a sustained increase in extracellular signal‐regulated kinase (ERK)‐1/2 phosphorylation. Furthermore, inhibition of neuronal nitric oxide synthase (nNOS), NO, and cGMP significantly blocked the inhibitory effects of relaxin on α‐SMA and Smad2 phosphorylation, while the NO inhibitor, l‐nitroarginine methyl ester (hydrochloride) (l‐NAME) significantly blocked the inhibitory actions of relaxin on collagen concentration in vivo. These findings suggest that relaxin signals through RXFP1, and a nNOS‐NO‐cGMP‐dependent pathway to inhibit Smad2 phosphorylation and interfere with TGF‐β1‐mediated renal myofibroblast differentiation and collagen production.—Mookerjee, I., Hewitson, T. D., Halls, M. L., Summers, R. J., Mathai, M. L., Bathgate, R. A. D., Tregear, G. W., Samuel, C. S. Relaxin inhibits renal myofibroblast differentiation via RXFP1, the nitric oxide pathway, and Smad2. FASEB J. 23, 1219–1229 (2009)

Hypoxia and hypoxia signaling in tissue repair and fibrosis.

Following injury, vascular damage results in the loss of perfusion and consequent low oxygen tension (hypoxia) which may be exacerbated by a rapid influx of inflammatory and mesenchymal cells with high metabolic demands for oxygen. Changes in systemic and cellular oxygen concentrations induce tightly regulated response pathways that attempt to restore oxygen supply to cells and modulate cell function in hypoxic conditions. Most of these responses occur through the induction of the transcription factor hypoxia-inducible factor-1 (HIF-1) which regulates many processes needed for tissue repair during ischemia in the damaged tissue. HIF-1 transcriptionally upregulates expression of metabolic proteins (GLUT-1), adhesion proteins (integrins), soluble growth factors (TGF-β and VEGF), and extracellular matrix components (type I collagen and fibronectin), which enhance the repair process. For these reasons, HIF-1 is viewed as a positive regulator of wound healing and a potential regulator of organ repair and tissue fibrosis. Understanding the complex role of hypoxia in the loss of function in scarring tissues and biology of chronic wound, and organ repair will aid in the development of pharmaceutical agents that can redress the detrimental outcomes often seen in repair and scarring.

Interstitial Myofibroblasts in Experimental Renal Infection and Scarring

We have examined the temporal and spatial distribution of myofibroblast-like cells, a phenotype with fibroblast and smooth muscle features, in an experimental model of renal infection. Escherichia coli organisms (10(5)) were inoculated directly into the renal cortex of Sprague-Dawley rats weighing 270 g. Saline was substituted in a control group. The animals were sacrificed at five time points up to day 24 (E. coli n = 8, controls n = 3 each interval). Myofibroblasts were identified by morphology and immunohistochemistry for alpha smooth muscle actin (alpha-SMA) and compared with staining for monocytes (ED-1), collagen III, and bromodeoxyuridine incorporation. Histological changes included a focal lesion in E. coli infected animals. Interstitial alpha-SMA staining was confined to spindle-shaped cells resembling myofibroblasts. The percent fractional area of alpha-SMA staining in the lesion increased from 0.12 +/- 0.09 at day 1 to 20.0 +/- 7.1 at day 3 (p < 0.005), decreasing progressively to 2.0 +/- 2.6 by day 24. This paralleled bromodeoxyuridine incorporation in myofibroblasts: 0.4 +/- 0.5 cells/0.25 mm2 at day 1, 105.0 +/- 36.3 at day 3, and 2.6 +/- 2.2 cells/0.25 mm2 at day 24. ED-1-positive cells increased from 374 +/- 200/0.25 mm2 at day 1 to 894 +/- 88 at day 3 (p < 0.01), declining to 230 +/- 108/0.25 mm2 by day 24. Intracellular collagen III and alpha-SMA stainings were colocalized at day 3. The fractional area of collagen III increased by day 24 (p < 0.05). In conclusion, myofibroblasts accumulate transiently during renal interstitial fibrosis and are derived at least in part from local proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Drugs of the future: the hormone relaxin

Abstract.The peptide hormone relaxin is emerging as a multi-functional factor in a broad range of target tissues including several non-reproductive organs, in addition to its historical role as a hormone of pregnancy. This review discusses the evidence that collectively demonstrates the many diverse and vital roles of relaxin: the homeostatic role of endogenous relaxin in mammalian pregnancy and ageing; its gender-related effects; the therapeutic effects of relaxin in the treatment of fibrosis, inflammation, cardioprotection, vasodilation and wound healing (angiogenesis) amongst other pathophysiological conditions, and its potential mechanism of action. Furthermore, translational issues using experimental models (to humans) and its use in various clinical trials, are described, each with important lessons for the design of future trials involving relaxin. The diverse physiological and pathological roles for relaxin have led to the search for its significance in humans and highlight its potential as a drug of the future.