G. Karakiulakis

The differential distribution of hyaluronic acid in the layers of human atheromatic aortas is associated with vascular smooth muscle cell proliferation and migration

Vascular smooth muscle cells (VSMC), under conditions of induced proliferation, similar to those involved in atherosclerosis, secrete an acidic glycan, 82% of which exhibits structural homology with hyaluronic acid (HA), has a molecular mass of 340 kDa (HA-340) and inhibits VSMC proliferation in vitro. In this study, the expression of glycans was investigated in human atheromatic aortas and evidence is presented that a HA molecule, similar to HA-340, is distinctly expressed in all aortic layers. The isolation of the glycans from human aortas was performed after homogenization of the individual aortic layers (atheromatic plaque, tunica intima, tunica media and tunica adventitia), by lipid extraction and extensive digestion with pronase and DNase. The total glycans were purified from the digestion products by gel filtration on Sephadex G-25 and fractionated on a Superose 6 column. Enzymatic treatment of the ensuing glycan fractions with all known glycosaminoglycan-degrading enzymes, followed by electrophoresis on polyacrylamide gradient gels and cellulose acetate membranes, revealed that, in addition to HA, the tunica intima and the atheromatic plaque also contained dermatan sulfate, while the tunica media and the tunica adventitia also contained chondroitin sulfates and heparan sulfate. The highest concentration of the human aorta HA was found in the tunica media, exhibiting a negative concentration gradient from the tunica media to the atheromatic plaque. Investigation of the biological function of the human aorta HA revealed that this molecule acts as a negative regulator on the PDGF-induced VSMC proliferation and as a positive regulator on the PDGF-induced VSMC migration. The differential expression of HA within the aortic layers correlates with the biological function attributed to this acidic glycan and associates it with key events in the progression of atherogenesis.

Decreased hyaluronan in airway smooth muscle cells from patients with asthma and COPD

Glycosaminoglycans (GAG) are essential extracellular matrix molecules which regulate tissue flexibility, a parameter that is reduced in airways of patients with asthma and chronic obstructive pulmonary disease (COPD). We investigated the expression of GAG and their metabolising enzymes in primary human airway smooth muscle cells (ASMC) obtained from healthy donors (controls) and patients with asthma or COPD. Total GAG synthesis was assessed by [3H]-glucosamine incorporation. GAG were isolated, purified, fractionated by electrophoresis and characterised using specific GAG-degrading enzymes. Secretion of hyaluronic acid (HA) by ASMC from patients with asthma or COPD was significantly decreased compared with controls. RT-PCR analysis and western blotting revealed that this decrease was associated with a significant reduction in the expression of HA synthase-1 and -2 and a significant increase of hyaluronidase-1. Furthermore, the expression of the HA receptor CD44 was significantly decreased, whereas the receptor for HA-mediated motility was not expressed in asthma or COPD. Our results indicate that there is a decreased expression of HA in asthma and COPD associated with a synergistic regulation of HA metabolising enzymes that may regulate the pathological airway remodelling in these lung diseases.

Extrinsic ageing in the human skin is associated with alterations in the expression of hyaluronic acid and its metabolizing enzymes

Abstract:  Extrinsic skin ageing or ‘photoageing’, as opposed to intrinsic skin ageing, is the result of exposure to external factors, mainly ultraviolet irradiation. Glycosaminoglycans (GAG) and particularly hyaluronic acid (HA) are major components of the cutaneous extracellular matrix involved in tissue repair. However, their involvement in extrinsic skin ageing remains elusive. In this study, we investigated the expression of HA and its metabolizing enzymes in photoexposed and photoprotected human skin tissue specimens, obtained from the same patient. Total GAG were isolated, characterized using specific GAG‐degrading enzymes and separated by electrophoresis on cellulose acetate membranes and polyacrylamide gels. Quantitation of HA in total GAG was performed using ELISA. Gene expression of hyaluronan synthases (HAS), hyaluronidases (HYAL) and HA receptors CD44 and receptor for HA‐mediated motility (RHAMM) was assessed by RT‐PCR. We detected a significant increase in the expression of HA, of lower molecular mass, in photoexposed skin as compared with photoprotected skin. This increase was associated with a significant decrease in the expression of HAS1 and an increase in the expression of HYAL1‐3. Furthermore, the expression of HA receptors CD44 and RHAMM was significantly downregulated in photoexposed as compared with photoprotected skin. These findings indicate that extrinsic skin ageing is characterized by distinct homoeostasis of HA. The elucidation of the role of HA homoeostasis in extrinsic skin ageing may offer an additional approach in handling cutaneous ageing.

Increased hyaluronic acid content in idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease characterised by elevated blood pressure in the pulmonary circulation. Initial vasoconstriction, proliferation of pulmonary arterial smooth muscle cells (PASMC) and increased deposition of extracellular matrix (ECM) contribute to pathological remodelling of pulmonary arterioles in IPAH. Glycosaminoglycans (GAGs), components of the ECM, control cellular proliferation and differentiation, but their expression in IPAH remains elusive. In the present study, GAG expression was investigated in the lungs of patients with IPAH or control transplant donors, and expression and localisation of GAG-metabolising enzymes were analysed in vivo and in vitro. A significant increase in the expression of hyaluronic acid (HA) was detected in IPAH lungs, associated with increased hyaluronan synthase (Has)1 and decreased hyaluronoglucosaminidase 1 gene expression, as assessed by quantitative RT-PCR and Western blotting. HAS1 protein localised to PASMC in vivo and increased HA deposition was observed in remodelled pulmonary arteries in IPAH. Transforming growth factor-β1, a profibrotic growth factor, led to increased HA secretion and HAS1 expression in primary PASMC. The results demonstrate an increased hyaluronic acid content in idiopathic pulmonary arterial hypertension lungs, associated with increased hyaluronan synthase 1 and decreased hyaluronoglucosaminidase 1 gene expression. Synergistic regulation of glycosaminoglycan-metabolising enzymes in favour of accumulation may, thus, regulate pathological vascular remodelling in idiopathic pulmonary arterial hypertension lungs.

Interleukin-6 and matrix metalloproteinase expression in the subretinal fluid during proliferative vitreoretinopathy: Correlation with extent, duration of RRD and PVR grade

PURPOSE To investigate interleukin (IL)-6 protein levels in the subretinal fluid (SRF) of patients with rhegmatogenous retinal detachment (RRD) complicated by proliferative vitreoretinopathy (PVR); to correlate the IL-6 levels with matrix metalloproteinases (MMP)-1, -2, -3, -8, -9 and tissue inhibitor of metalloproteinases (TIMP)-1 with respect to RRD extent, duration and PVR grade. METHODS Thirty-one SRF samples from 31 eyes of 31 patients with RRD complicated with PVR and five SRF samples from five eyes of five patients suffering from RRD not complicated with PVR were collected during treatment by scleral buckling. Enzyme-Linked Immunosorbent Assay was employed for the measurement of IL-6, MMP-1, -3, -8 and TIMP-1 levels while the enzymatic activity of MMP-2 and MMP-9 was assessed by gelatin zymography. RESULTS Protein levels of IL-6 (p=0.050), MMP-1 (p=0.001), MMP-3 (p=0.005), MMP-8 (p=0.003), TIMP-1 (p=0.001) as well as enzymatic activity of proMMP-2 (p=0.001), MMP-2 (p=0.023) and MMP-9 (p=0.015), were significantly higher in the SRF of PVR patients compared to controls. IL-6 levels correlated significantly with TIMP-1 (r=0.528, p=0.035). Regarding clinical parameters of the detachment, IL-6 levels correlated with RRD extent (r=0.592, p=0.016), but not with RRD duration (p=0.857) and PVR grade (p=0.594). Regression analysis revealed positive correlations between IL-6 and MMP-2. CONCLUSIONS There was a significant correlation between IL-6 and TIMP-1 levels in the SRF of PVR patients. The findings of this study are in agreement with relevant studies concerning IL-6 involvement in the modulation of MMP expression and are indicative of IL-6 and MMP activity during PVR, mainly that of MMP-2 and TIMP-1.

Expression of proteoglycans and glycosaminoglycans in angiofibroma and fibrous plaque skin lesions from patients with tuberous sclerosis.

Tuberous sclerosis complex (TSC) is a disorder of cell lineage, migration, proliferation and differentiation, characterized by the development of widespread benign hamartomas, which are particularly evident in hamartomatous lesions of the skin. The aim of this study was to investigate differences in gene expression of certain proteoglycans (PGs) and to characterize glycosaminoglycans (GAGs) in tissue specimens of normal skin, fibrous plaques and angiofibromas from patients with TSC. The expression of PG mRNA was determined by semiquantitative RT-PCR analysis. Total GAGs were isolated from tissue specimens after lipid extraction and extensive digestion with Pronase and DNase and characterized by treatment with GAG-degrading enzymes followed by electrophoresis on polyacrylamide gradient gels and cellulose acetate membranes. Normal skin specimens express versican, decorin and aggrecan and contain hyaluronic acid and dermatan sulphate. In angiofibroma specimens aggrecan is not expressed while versican splice variant with two EGF-like domains and decorin are downregulated. Furthermore, angiofibromas differ from normal skin in that they additionally contain keratan, heparan and chondroitin sulphates and do not contain dermatan sulphate. In fibrous plaque specimens gene expression of PGs was similar to that in normal skin, but with respect to GAGs, they contained a single acidic glycan population that did not share common structural features with known GAGs. The variations of the above ECM molecules between normal and TSC skin may be attributed to TSC-related mutations and, overall, support the TSC-associated pathological manifestations of cell migration, proliferation and differentiation.

The Effect of Celecoxib Administration on the Healing and Neovascularization of Colonic Anastomosis in Rats

ABSTRACT Background: The aim of this experimental study was to investigate whether the perioperative administration of the selective cyclooxygenase-2 inhibitor celecoxib affects the angiogenetic process and the healing of colonic anastomoses. Methods: Seventy-two male Wistar rats underwent colonic resection and anastomosis. Celecoxib (10 mg/kg/day—celecoxib group) or placebo (control group) was administered perioperatively. Rats of both groups were sacrificed on either the third or the seventh postoperative day and bursting pressures of the anastomoses were measured. Gelatine-degrading matrix metalloproteinases (MMPs) were identified with gelatine zymography, and proMMP-2 and vascular endothelial growth factor (VEGF) levels from both anastomotic site and tissue adjacent to the anastomosis were evaluated. Histologic evaluation of microvessels was performed by immunohistochemistry using an anti-CD34 monoclonal antibody. Results: Celecoxib did not significantly decrease anastomotic bursting pressures. Gelatin zymography revealed the presence of MMP-2, proMMP-2, and proMMP-9. MMP concentration was higher at the anastomotic tissue as compared with tissue distant to the anastomosis. Celecoxib resulted in a significant reduction in proMMP-2 levels at the anastomosis at both third and seventh postoperative day. VEGF levels from the anastomotic tissue were also found lower in the celecoxib group. Histological examination showed a celecoxib-induced reduction of newly formed CD34-stained vessels. Conclusions: Although the perioperative administration of celecoxib resulted in suppression of angiogenesis in the newly formed anastomoses, bursting pressures remained unaffected and subsequently safety was not compromised.

Nodular basal cell carcinoma is associated with increased hyaluronan homeostasis

Background  Basal cell carcinoma (BCC) is one of the most frequent forms of malignancy in humans. Although BCC is a tumour of low degree of malignancy, if left untreated, it can be locally aggressive, eat away at tissues and cause ulceration. Nodular is the most common subtype of BCC (>50%). Although apparently non‐invasive, micronodular, a certain subgroup of nodular, is likely to recur. Glycosaminoglycans (GAGs), such as hyaluronic acid (HA), are extracellular matrix molecules of high importance in malignant transformation, metastasis and other complex remodelling processes.

Furosemide modifies heart hypertrophy and glycosaminoglycan myocardium content in a rat model of neurogenic hypertension

Hypertension is a major risk factor for atherogenesis and heart hypertrophy, both of which are associated with specific morphological and functional changes of the myocardium. Glycosaminoglycans (GAGs) are complex molecules involved both in tissue morphology and function. In the present study, we investigated the effects of neurogenic hypertension and subsequent antihypertensive treatment with furosemide, on heart hypertrophy and the content of GAGs in the myocardium. Neurogenic hypertension was achieved in male Wistar rats by bilateral aortic denervation (bAD). At days 2, 7 and 15 after surgery, animals were sacrificed and the hearts were dissected away, weighted, and homogenized. Total GAGs were assessed by measuring the uronic acid content colorimetrically and individual GAGs were isolated and characterized by enzymatic treatment, with GAG-degrading enzymes, using electrophoresis on polyacrylamide gradient gels and cellulose acetate membranes. In bAD-animals blood pressure, blood pressure lability, heart rate and heart weight were significantly increased 15 days postoperatively. These effects were prevented by treatment with furosemide. Major GAGs identified in the heart were chondroitin sulphates, heparin (H), heparan sulphate (HS) and hyaluronic acid. The content of uronic and the relative content of H and HS in the heart in bAD animals significantly decreased from day 2 to day 15 postoperatively. Furosemide prevented the bAD induced decrease in GAG content. Considering that H and HS are potent inhibitors of cardiomyocyte hypertrophy, our results indicate that heart hypertrophy induced by neurogenic hypertension may be associated with decreases in the relative content of heparin and heparan sulphate in the heart.