Cellular and molecular facets of keratinocyte reepithelization during wound healing

Cutaneous wound healing is a highly coordinated physiological process that rapidly and efficiently restores skin integrity. Reepithelization is a crucial step during wound healing, which involves migration and proliferation of keratinocytes to cover the denuded dermal surface. Recent advances in wound biology clarified the molecular pathways governing keratinocyte reepithelization at wound sites. These new findings point towards novel therapeutic targets and provide suitable methods to promote faster tissue regeneration in vivo.     

Genetic and dietary interactions in the regulation of HMG-CoA reductase gene expression.

Inbred strains of mice exhibit large genetic variations in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. A tissue-specific genetic variation between the strains BALB/c and C57BL/6, resulting in about 5-fold higher levels in hepatic reductase activity in strain C57BL/6, was examined in detail. The activity difference between these two strains could be explained entirely by differences in hepatic reductase mRNA levels. In genetic crosses, the variation segregated as a single major Mendelian element. Surprisingly, the mode of inheritance was recessive since F1 mice exhibited the BALB/c levels of enzyme activity. Despite the fact that the rates of hepatic sterol synthesis also differed between the strains by a factor of about five, the altered hepatic reductase expression did not significantly influence plasma lipoprotein levels. The response to a high cholesterol, high fat diet between the strains was remarkably different. Thus, in BALB/c mice, both hepatic reductase activity and mRNA levels were affected only slightly, if at all, by cholesterol feeding, while in strain C57BL/6 mice both were reduced more than 10-fold by cholesterol feeding. Several lines of evidence, including analysis of cis-acting regulatory elements, the nonadditive mode of inheritance, and genetic studies of the HMG-CoA reductase gene locus on mouse chromosome 13, support the possibility that the variation in reductase expression is not due to a mutation of the structural gene but, rather, is determined by a trans-acting factor controlling reductase mRNA levels. The variation provides a striking example, at the molecular level, of the importance of dietary-genetic interactions in the control of cholesterol metabolism.     

Flexor tendon wound healing in vitro: lactate up-regulation of TGF-beta expression and functional activity

Flexor tendon wound healing in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. These adhesions can significantly alter tendon gliding and ultimately hand function. Lactate and transforming growth factor-beta (TGF-beta) are two important mediators of wound healing that have been demonstrated to independently increase collagen production by cells of the tendon sheath, epitenon, and endotenon. This study examined the effects of lactate on TGF-beta peptide and receptor production by flexor tendon cells. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. Cell cultures were supplemented with 50 mM lactate, and the expression of three TGF-beta peptide isoforms (beta1, beta2, and beta3) and three receptor isoforms (R1, R2, and R3) was quantified with enzyme-linked immunosorbent assays. TGF-beta functional activity was also assessed with the addition of tendon cell conditioned media to mink lung epithelial cells transfected with a luciferase reporter gene expression construct responsive to TGF-beta. Supplementation of the cell culture medium with lactate significantly (p < 0.05) increased the expression of all TGF-beta peptide and receptor isoforms in all three cell lines. Tendon sheath fibroblasts exhibited the greatest increases in beta1 and beta2 peptide isoform expression (30 and 23 percent, respectively), whereas endotenon tenocytes demonstrated the greatest increase in beta3 peptide expression (32 percent). Epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression (17 and 19 percent, respectively). All three tendon cell types demonstrated significant (p < 0.05) increases in TGF-beta functional activity when exposed to lactate. Epitenon tenocytes demonstrated the greatest increase in activity (>4 times control values), whereas tendon sheath fibroblasts demonstrated the highest overall levels of total TGF-beta functional activity. Lactate significantly increased TGF-beta peptide (beta1, beta2, and beta3) expression, receptor (R1, R2, and R3) expression, and functional activity, suggesting a common pathway regulating tendon cell collagen production. Modulation of lactate and TGF-beta levels may provide a means of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.     

Phenotypic changes and possible angiogenic roles of pericytes during wound healing in the mouse skin

Pericytes (PCs) are attracting increasing attention as a crucial target for anti-angiogenic therapy. In this study, we sought to determine the functional significance of PCs during angiogenesis by using a skin wound healing model in which different angiogenic stages are identifiable. Angiogenesis was first observed on Day 3 after wounding and increased greatly on Day 5. On Day 5, the leading edge of the regenerating vessels (vascular advancing front; VAF) appeared to be composed of immature vessels, and was further divided into "tip" and "following" regions according to maturational differences. PCs distributed in regenerating vessels showed phenotypic differences according to different regions. PCs that expressed PDGFR-β alone and lacked vascular basement membrane (BM) were predominant in the tip region of the VAF, while PCs that expressed both PDGFR-β and NG2 with their BM coating were numerous in the following regions toward the rear of the VAF. Moreover, PCs in the VAF expressed VEGF-A and associated with most proliferating endothelial cells (ECs). VEGF-A expression of PCs and the proliferating ECs totally disappeared in the region toward the rear of the VAF. We conclude that PCs can differ in their phenotype according to the stage of angiogenesis during wound healing. They may promote angiogenesis at the initial stage but might in turn stabilize the newly formed vessels at the later stage.     

Plasma membrane sphingomyelin and the regulation of HMG-CoA reductase activity and cholesterol biosynthesis in cell cultures

We have examined the mechanism of the inhibition of cholesterol synthesis in cells treated with exogenous sphingomyelinase. Treatment of rat intestinal epithelial cells (IEC-6), human skin fibroblasts (GM-43), and human hepatoma (HepG2) cells in culture with sphingomyelinase resulted in a concentration- and time-dependent inhibition of the activity of HMG-CoA reductase, a key regulatory enzyme in cholesterol biosynthesis. The following observations were obtained with IEC-6 cells. Free fatty acid synthesis or general cellular protein synthesis was unaffected by the addition of sphingomyelinase. Addition of sphingomyelinase to the in vitro reductase assay had no effect on activity, suggesting that an intact cell system is required for the action of sphingomyelinase. The products of sphingomyelin hydrolysis, e.g., ceramide and phosphocholine, had no effect on reductase activity. Sphingosine, a further product of ceramide metabolism, caused a stimulation of reductase activity. Examination of the incorporation of [3H]acetate into the nonsaponifiable lipid fractions in the presence of sphingomyelinase showed no changes in the percent distribution of radioactivity in the post-mevalonate intermediates of the cholesterol biosynthetic pathway, but there was increased radioactivity associated with the polar sterol fraction. Pretreatment of cells with ketoconazole, a known inhibitor of oxysterol formation, prevented the inhibition of reductase activity by sphingomyelinase and decreased the incorporation of [3H]acetate in the polar sterol fraction. Ketoconazole had no effect on exogenous sphingomyelinase activity in vitro in the presence or absence of cells. Endogenous sphingomyelinase activity was also unaffected by ketoconazole. Addition of inhibitors of endogenous sphingomyelinase activity, e.g., chlorpromazine, desipramine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), to the culture medium caused a dose-dependent stimulation of reductase activity. However, these agents had no effect on the inhibition of reductase activity by exogenous sphingomyelinase. Treatment of cells with small unilamellar vesicles of dioleyl phosphatidylcholine or high density lipoprotein3 resulted in increased efflux of cholesterol and stimulation of reductase activity. Under similar conditions, the inhibitory effect of exogenous sphingomyelinase on reductase activity was prevented by incubation with small unilamellar vesicles of phosphatidylcholine or high density lipoprotein. These results support the hypothesis that alteration of the ratio of sphingomyelin:cholesterol in the plasma membrane plays a modulatory role on the flow of membrane cholesterol to a site where it may be converted to a putative regulatory molecule, possibly an oxysterol.     

Cytochrome P450 epoxygenases and vascular tone: novel role for HMG-CoA reductase inhibitors in the regulation of CYP 2C expression

Over the last 10 years it has become increasingly clear that cytochrome P450 (CYP) enzymes expressed within endothelial and vascular smooth muscle cells play a crucial role in the modulation of vascular homeostasis. There is strong evidence suggesting that the activation of a CYP 2C epoxygenase in endothelial cells is an essential step in nitric oxide (NO)- and prostacyclin (PGI(2))-independent vasodilatation of several vascular beds, particularly in the heart and kidney. Moreover, CYP epoxygenase products as well as CYP-derived reactive oxygen species are intracellular signal transduction molecules involved in several signaling cascades affecting numerous cellular processes, including vascular cell proliferation and angiogenesis. Various pharmacological compounds enhance vascular CYP 2C expression. One group of substances which highlight the possible effects of CYP induction in endothelial cells on vascular function are the HMG-CoA reductase inhibitors (statins). Cerivastatin and fluvastatin increase CYP 2C mRNA and protein in native and cultured endothelial cells, and enhance the bradykinin-induced NO/PGI(2)-independent relaxation of arterial segments as well as the generation of reactive oxygen species. However, statins also increase the expression of the endothelial NO synthase by approximately twofold. As a consequence, the probability that NO and reactive oxygen species react to generate peroxynitrite is increased and the treatment of vascular segments with statins resulted in enhanced protein tyrosine nitration. These data highlight the role played by CYP 2C in vascular homeostasis and its potential regulation by cardiovascular drugs.     

The role of bovine lipoproteins in the regulation of steroidogenesis and HMG-CoA reductase in bovine adrenocortical cells.

The sources of cholesterol for steroid hormone production were examined using bovine adrenocortical (BAC) cells in primary culture. The experiments were designed to determine the effects of lipoproteins on cortisol production and the level of BAC cell 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Most studies on BAC cell lipoprotein requirements have been conducted using human low-density lipoprotein (hHDL); none have used the homologous bovine lipoproteins. BAC cells treated with corticotropin (ACTH) in a medium devoid of lipoproteins increased and maintained cortisol production 7- to 20-fold above basal levels. Under such conditions ACTH also increased the rate of HMG-CoA reductase activity. Inhibition of HMG-CoA reductase with mevinolin inhibited cortisol production by 85%, indicating that the cells were using cholesterol synthesized de novo for steroid production. Cortisol production was increased almost 40-fold above basal levels if hLDL (100 micrograms/ml) was included in the incubation medium. Human LDL also suppressed the levels of HMG-CoA reductase in a concentration-dependent fashion. Human HDL was without effect on either BAC cell steroidogenesis of HMG-CoA reductase. Addition of bovine LDL (bLDL) to the incubation medium also caused an increase in cortisol production and inhibited cholesterol synthesis. By contrast to hHDL, bHDL (100 micrograms/ml) increased the ability of BAC cells to produce cortisol production. Bovine HDL (bHDL) also was able to decrease HMG-CoA reductase, but not to the extent caused by hLDL or bLDL. These data demonstrate that bovine adrenal cells can use bHDL as a source of cholesterol for steroid hormone production. These findings may be of particular importance when one considers that in vivo, the bHDL content of bovine serum greatly surpasses the level of bLDL.     

The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: implications for wound healing

Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin alpha3beta1- and/or alpha6beta4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-beta1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.     

Resveratrol attenuates the expression of HMG-CoA reductase mRNA in hamsters

We investigated the hypolipidemic effect of resveratrol focused on the mRNA expression and hepatic HMG-CoA reductase (HMGR) activity in hamsters fed a high-fat diet. Male Syrian Golden hamsters were fed a high-fat diet containing 0.025% fenofibrate or 0.025% resveratrol for 8 weeks. The concentrations of serum total cholesterol and triglyceride were significantly lower in the resveratrol-fed group than in the control group. The resveratrol contained diet significantly decreased Apo B, Lp(a), and cholesterol-ester-transport protein (CETP) concentrations, but increased Apo A-I levels and the Apo A-I/Apo B ratio. The contents of cholesterol and triglyceride in hepatic tissue were significantly lower in the resveratrol group than in the control group. Real-time PCR analysis revealed that HMGR mRNA expression was significantly lower in the resveratrol group than in the control group. These results indicate that dietary resveratrol reduces serum cholesterol by down-regulating hepatic HMGR mRNA expression in hamsters fed a high-fat diet.     

Crystal structure of the catalytic portion of human HMG-CoA reductase: insights into regulation of activity and catalysis

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the formation of mevalonate, the committed step in the biosynthesis of sterols and isoprenoids. The activity of HMGR is controlled through synthesis, degradation and phosphorylation to maintain the concentration of mevalonate-derived products. In addition to the physiological regulation of HMGR, the human enzyme has been targeted successfully by drugs in the clinical treatment of high serum cholesterol levels. Three crystal structures of the catalytic portion of human HMGR in complexes with HMG-CoA, with HMG and CoA, and with HMG, CoA and NADP(+), provide a detailed view of the enzyme active site. Catalytic portions of human HMGR form tight tetramers. The crystal structure explains the influence of the enzyme's oligomeric state on the activity and suggests a mechanism for cholesterol sensing. The active site architecture of human HMGR is different from that of bacterial HMGR; this may explain why binding of HMGR inhibitors to bacterial HMGRs has not been reported.     

Cell and molecular regulation of endothelin-1 production during hepatic wound healing

During hepatic wound healing, activation of key effectors of the wounding response known as stellate cells leads to a multitude of pathological processes, including increased production of endothelin-1 (ET-1). This latter process has been linked to enhanced expression of endothelin-converting enzyme-1 (ECE-1, the enzyme that converts precursor ET-1 to the mature peptide) in activated stellate cells. Herein, we demonstrate up-regulation of 56- and 62-kDa ECE-1 3'-untranslated region (UTR) mRNA binding proteins in stellate cells after liver injury and stellate cell activation. Binding of these proteins was localized to a CC-rich region in the proximal ECE-1 3' UTR base pairs (the 56-kDa protein) and to a region between 60 and 193 base pairs in the ECE-1 3' UTR mRNA (62 kDa). A functional role for the 3' UTR mRNA/protein interaction was established in a series of reporter assays. Additionally, transforming growth factor-beta1, a cytokine integral to wound healing, stimulated ET-1 production. This effect was due to ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells, which in turn was a result of de novo synthesis of the identified 56- and 62-kDa ECE-1 3' UTR mRNA binding proteins. These data indicate that liver injury and the hepatic wound healing response lead to ECE-1 mRNA stabilization in stellate cells via binding of 56- and 62-kDa proteins, which in turn are regulated by transforming growth factor-beta. The possibility that the same or similar regulatory events are present in other forms of wound healing is raised.     

Phosphatase activity of blood leukocytes and wound exudate during the experimental healing of an aseptic wound

Cytochemical investigations of plain aseptic wounds simulated in 110 Wistar rats revealed a clear-cut dependence between the variations in the activity of alkaline and acid phosphatases in neutrophilic leukocytes, monocytes and lymphocytes of the blood and wound exudate and the stage of the healing process. Elevated activity of the blood alkaline phosphatase correlated with the term of inflammation phase.     

Efficacy of l-proline administration on the early responses during cutaneous wound healing in rats

Proline (Pro) plays a versatile role in cell metabolism and physiology. Pro and hydroxypro are major imino acids present in collagen, an important connective tissue protein, essential for wound healing, which is a primary response to tissue injury. This study explains the role of l-pro on cutaneous wound healing in rats when administered both topically and orally. Open excision wounds were made on the back of rats, and 200 μl (200 mg) of pro was administered topically and orally once daily to the experimental rats until the wounds healed completely. The control wounds were left untreated. Granulation tissues formed were removed after day 4 and 8 of post excision wounding, and biochemical parameters such as total protein, collagen, hexosamine, and uronic acid were estimated. Levels of enzymatic and non-enzymatic antioxidants such as catalase, superoxide dismutase, glutathione peroxidase, ascorbic acid, and reduced glutathione were evaluated along with lipid peroxides in the granulation tissues. Tensile strength and period of epithelialization were also measured. It was observed that the treated wounds healed very fast as evidenced by augmented rates of epithelialization and wound contraction, which was also confirmed by histological examinations. The results strappingly authenticate the beneficial effects of the topical administration of l-proline in the acceleration of wound healing than the oral administration and control.