George Karakiulakis

Corticosteroids inhibit the expression of the vascular endothelial growth factor gene in human vascular smooth muscle cells

The vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and enhances vascular permeability and edemagenesis. VEGF is also a major regulator of angiogenesis and may be a key target for inhibiting angiogenesis in angiogenesis-associated diseases. Among the extensively studied angiostatic compounds are several corticosteroids when used alone or in combination with heparin. In this study we present evidence for an additional mechanism of action of hydrocortisone, cortisone and dexamethasone in inhibiting edemagenesis or angiogenesis. In cultures of aortic human vascular smooth muscle cells these corticosteroids (1 x 10(-8) to 1 x 10(-12) M) abolished the platelet-derived growth factor-induced (PDGF) expression of the VEGF gene in a dose-dependent manner. In contrast, two precursors of corticosteroids, desoxycorticosterone or pregnenolone, did not affect PDGF-induced VEGF expression. Our findings indicate that the capacity of corticosteroids to reduce edema or to prevent new blood vessel formation may be attributed, at least in part to the ability of these agents to abolish the expression of VEGF.

Hyaluronic acid: A key molecule in skin aging

Skin aging is a multifactorial process consisting of two distinct and independent mechanisms: intrinsic and extrinsic aging. Youthful skin retains its turgor, resilience and pliability, among others, due to its high content of water. Daily external injury, in addition to the normal process of aging, causes loss of moisture. The key molecule involved in skin moisture is hyaluronic acid (HA) that has unique capacity in retaining water. There are multiple sites for the control of HA synthesis, deposition, cell and protein association and degradation, reflecting the complexity of HA metabolism. The enzymes that synthesize or catabolize HA and HA receptors responsible for many of the functions of HA are all multigene families with distinct patterns of tissue expression. Understanding the metabolism of HA in the different layers of the skin and the interactions of HA with other skin components will facilitate the ability to modulate skin moisture in a rational manner.

Hypoxia modulates the effects of transforming growth factor-β isoforms on matrix-formation by primary human lung fibroblasts

Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-beta (TGF-beta) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-beta on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-beta1, TGF-beta2 or TGF-beta3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-betas significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-beta and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-betas-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.

Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo.AimTo determine the in vitro effect of nintedanib on primary human lung fibroblasts. Methods: Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen.ResultsIPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib.ConclusionOur data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug’s anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.

Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

Hypoxia is associated with extracellular matrix remodeling in several inflammatory lung diseases, such as fibrosis, chronic obstructive pulmonary disease, and asthma. In a human cell culture model, we assessed whether extracellular matrix modification by hypoxia and platelet-derived growth factor (PDGF) involves the action of matrix metalloproteinases (MMPs) and thereby affects cell proliferation. Expression of MMP and its activity were assessed by zymography and enzyme-linked immunosorbent assay in human lung fibroblasts and pulmonary vascular smooth muscle cells (VSMCs), and synthesis of soluble collagen type I was assessed by enzyme-linked immunosorbent assay. In both cell types, hypoxia up-regulated the expression of MMP-1, -2, and -9 precursors without subsequent activation. MMP-13 was increased by hypoxia only in fibroblasts. PDGF-BB inhibited the synthesis and secretion of all hypoxia-dependent MMP via Erk1/2 mitogen-activated protein (MAP) kinase activation. Hypoxia and PDGF-BB induced synthesis of soluble collagen type I via Erk1/2 and p38 MAP kinase. Hypoxia-induced cell proliferation was blocked by antibodies to PDGF-BB or by inhibition of Erk1/2 but not by the inhibition of MMP or p38 MAP kinase in fibroblasts. In VSMCs, hypoxia-induced proliferation involved Erk1/2 and p38 MAP kinases and was further increased by fibroblast-conditioned medium or soluble collagen type I via Erk1/2. In conclusion, hypoxia controls tissue remodeling and proliferation in a cell type-specific manner. Furthermore, fibroblasts may affect proliferation of VSMC indirectly by inducing the synthesis of soluble collagen type I.

Angiogenic Growth Factors and their Inhibitors in Diabetic Retinopathy

Diabetic retinopathy is considered one of the vision-threatening diseases among working-age population. The pathogenesis of the disease is regarded multifactorial and complex: capillary basement membrane thickening, loss of pericytes, microaneuryms, loss of endothelial cells, blood retinal barrier breakdown and other anatomic lesions might contribute to macular edema and/or neovascularization the two major and sight threatening complications of diabetic retinopathy. A number of proangiogenic, angiogenic and antiangiogenic factors are involved in the pathogenesis and progression of diabetic retinal disease, Vascular Endothelial Growth Factor (VEGF) being one of the most important. Other growth factors, which are known to participate in the pathogenesis of the disease, are: Platelet Derived Growth Factor (PDGF), Fibroblast Growth Factor (FGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor (TGF), Placental Endothelial Cell Growth Factor (PlGF), Connective Tissue Growth Factor (CTGF). Other molecules that are involved in the disease mechanisms are: intergrins, angiopoietins, protein kinase C (PKC), ephrins, interleukins, leptin, angiotensin, monocyte chemotactic protein (MCP), vascular cell adhesion molecule (VCAM), tissue plasminogen activator (TPA), and extracellular matrix metalloproteinases (ECM-MMPs). However, the intraocular concentration of angiogenic factors is counterbalanced by the ocular synthesis of several antioangiogenic factors such as pigment epithelial derived factor (PEDF), angiostatin, endostatin, thrombospondin, steroids, atrial natriuretic peptide (ANP), inteferon, aptamer, monoclonal antibodies, VEGF receptor blocker, VEGF gene suppressors, intracellular signal transduction inhibitors, and extracellular matrix antagonists. Growth stimulation or inhibition by these factors depends on the state of development and differentiation of the target tissue. The mechanisms of angiogenesis factor action are very different and most factors are multipotential; they stimulate proliferation or differentiation of endothelial cells. This review attempts to briefly outline the knowledge about peptide growth factor involvement in diabetic retinopathy. Further ongoing research may provide better understanding of molecular mechanisms, disease pathogenesis and therapeutic interactions.

The 'sweet' and 'bitter' involvement of glycosaminoglycans in lung diseases: pharmacotherapeutic relevance

The extracellular matrix (ECM) plays a significant role in the structure and function of the lung. The ECM is a three‐dimensional fibre mesh, comprised of various interconnected and intercalated macromolecules, among which are the glycosaminoglycans (GAG). GAG are long, linear and highly charged, heterogeneous polysaccharides that are composed of a variable number of repeating disaccharide units (macromolecular sugars) and most of them, as their name implies, have a sweet taste. In the lung, GAG support the structure of the interstitium, the subepithelial tissue and the bronchial walls, and are secreted in the airway secretions. Besides maintaining lung tissue structure, GAG also play an important role in lung function as they regulate hydration and water homeostasis, modulate the inflammatory response and influence lung tissue repair and remodelling. However, depending on their size and/or degree of sulphation, and their immobilization or solubilization in the ECM, specific GAG in the lung either live up to their sweet taste/name, supporting normal lung physiology, or they are associated to ‘bitter’ effects, related to lung pathology. The present review discusses the biological role of GAG in the lung as well as the involvement of these molecules in various respiratory diseases. Given the great structural diversity of GAG, understanding the changes in GAG expression that occur in lung diseases may lead to novel targets for pharmacological intervention in order to prevent and/or to treat a range of lung diseases.

Vitreous and serum levels of vascular endothelial growth factor and platelet-derived growth factor and their correlation in patients with non-proliferative diabetic retinopathy and clinically significant macula oedema.

Purpose:  To investigate possible correlations between vitreous and/or serum levels of platelet‐derived growth factor isoforms (PDGF‐AA, ‐AB and ‐BB) with parameters associated with non‐proliferative diabetic retinopathy (NPDR) and clinically significant macula oedema (CSMO); to compare the results to relevant results regarding vascular endothelial growth factor (VEGF), which is an established growth factor affecting NPDR.

Vitreous and Serum Levels of Platelet-Derived Growth Factor and Their Correlation in Patients with Proliferative Diabetic Retinopathy

Purpose: We investigated possible correlations between vitreous and/or serum levels of platelet derived growth factor isoforms (PDGF-AA, -AB, -BB) with parameters associated with proliferative diabetic retinopathy (PDR), and compared the results to vascular endothelial growth factor (VEGF), which is an established growth factor affecting PDR. Methods: Thirty-one patients with PDR and 15 non-diabetic patients were included in the study. Vitreous and serum samples were obtained during vitrectomy. PDGF-AA, -AB, and -BB, as well as VEGF, were measured by enzyme-linked immunosorbent assay. Results: PDGF-AA, -AB, -BB, and VEGF were all expressed in serum and vitreous of controls and patients with PDR. The levels of all PDGF isoforms and VEGF in vitreous were significantly increased in the PDR group, as compared to controls. No such differences were evident in serum. PDGF-AA and PDGF-BB correlated significantly to the severity but not the activity of PDR. PDGF-AB and -BB were significantly lower in vitreous of patients with pre-performed complete panretinal photocoagulation (PRP) as compared to incomplete or without PRP. PDGF did not correlate significantly to fibrovascular tissue, on the disc or elsewhere, to long-standing vitreous hemorrhage, to tractional retinal detachment, or to posterior vitreous detachment. PDGF or VEGF in vitreous or serum of PDR patients did not correlate with the serum levels of HbA1C. There was no correlation between the vitreous and serum levels of VEGF or PDGF in patients with PDR. Conclusions: It appears that, in addition to VEGF, PDGF-AA, -AB, and -BB in the vitreous are also correlated with PDR.

Losartan inhibits the angiotensin II-induced modifications on fibrinolysis and matrix deposition by primary human vascular smooth muscle cells.

Disorders in the fibrinolytic and renin-angiotensin-aldosterone systems and excessive extracellular matrix (ECM) deposition are determinant factors in several pathologic manifestations of vascular and cardiac tissue. We used primary human vascular smooth muscle cells (VSMC) and studied the effects of losartan on angiotensin II (Ang II)–mediated (a) DNA synthesis, (b) secretion of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), (c) secretion of matrix metalloprotease-2 (MMP-2) activity and tissue inhibitors of MMPs (TIMPs), and (d) synthesis of glycosaminoglycans. VSMC cultures, established from human pulmonary arteries, were treated with Ang II (0.1 nM–1 &mgr;M) and/or losartan (0.1–10 &mgr;M), for 24 or 48 h. DNA synthesis was assessed by incorporation of 3 H-thymidine into VSMC, secreted tPA, PAI-1, and TIMPs by enzyme-linked immunosorbent assay, MMP-2 activity by gelatin zymography, and glycosaminoglycan synthesis by 3 H-glucosamine incorporation. Ang II (1 &mgr;M) enhanced DNA synthesis and secretion of PAI-1 and glycosaminoglycans and decreased secretion of MMP-2 activity and tPA, whereas it had no effect on secretion of TIMPs and glycosaminoglycans associated with cell layers. The Ang II-mediated effects were reversed by losartan, in a concentration-dependent manner. Losartan alone increased secretion of tPA, MMP-2 activity, and TIMPs and decreased secretion of PAI-1. These results indicate that AT 1 receptors are implicated in Ang II-mediated disorders of fibrinolysis and excessive ECM deposition by VSMC.