Cytoskeletal changes induced by excess extracellular matrix impair endothelial cell replication.

Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachussets 02114, USA.
Diabetologia (Impact Factor: 6.49). 09/1997; 40(8):879-86. DOI: 10.1007/s001250050763
Source: PubMed

ABSTRACT Thickening of basement membranes is an early and characteristic feature of diabetic vessels, but its consequences on the properties of vascular cells remain undefined. We investigated whether and how excess extracellular matrix (ECM) alters the replication of vascular endothelial cells in vitro. To test the effects of endogenous excess matrix, human umbilical vein endothelial cells (HUVEC) were plated on ECM produced under culture conditions (high ambient glucose) that increase ECM synthesis. Four of six HUVEC isolates plated on such ECM yielded a lower cell number (68 +/- 18%) than cells plated on control ECM. Growth inhibition was observed in HUVEC cultured on elevated concentrations (10 and 50 microg/ml) of exogenous fibronectin, when compared with HUVEC plated on tissue culture plastic or 0.25, 1.0, and 5.0 microg/ml fibronectin; the decreased replication was attributable to delayed transit through the G1 phase of the cell cycle. HUVEC grown on both 1 and 10 microg/ml fibronectin exhibited a modest upregulation of the fibronectin-specific integrin receptor alpha5beta1, and increased attachment to fibronectin substratum. However, unique to the HUVEC plated on growth-inhibitory concentrations of fibronectin was a redistribution in situ of integrins and vinculin to form more numerous focal adhesions, and an increased polymerization of cytoskeletal actin to form stress fibers. Concentrations (0.01 microg/ml) of cytochalasin D intended to prevent excess actin polymerization prevented the growth inhibition. Thus, excess ECM hampers endothelial cell replication in vitro through increased cell-ECM adhesion and attendant cytoskeletal rearrangements. These phenotypic changes provide probes to test whether cell-ECM interactions are altered in diabetic vessels in a direction that may compromise orderly endothelial cell renewal and its antithrombogenic function.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Pericyte survival in diabetic retinopathy depends also on interactions with extracellular matrix (ECM) proteins, which are degraded by matrix metalloproteinases (MMP). Elevated glucose influences ECM turnover, through expression of MMP and their tissue inhibitors, TIMP. We reported on reduced pericyte adhesion to high glucose-conditioned ECM and correction by thiamine. We aimed at verifying the effects of thiamine and benfotiamine on MMP-2, MMP-9 and TIMP expression and activity in human vascular cells with high glucose. In HRP, MMP-2 activity, though not expression, increased with high glucose and decreased with thiamine and benfotiamine; TIMP-1 expression increased with high glucose plus thiamine and benfotiamine; MMP-9 was not expressed. In EC, MMP-9 and MMP-2 expression and activity increased with high glucose, but thiamine and benfotiamine had no effects; TIMP-1 expression was unchanged. Neither glucose nor thiamine modified TIMP-2 and TIMP-3 expression. TIMP-1 concentrations did not change in either HRP or EC. High glucose imbalances MMP/TIMP regulation, leading to increased ECM turnover. Thiamine and benfotiamine correct the increase in MMP-2 activity due to high glucose in HRP, while increasing TIMP-1.
    Acta Diabetologica 05/2009; 47(2):105-11. · 4.63 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Hyperglycemia is the most prevalent characteristic of diabetes and plays a central role in mediating adverse effects on vascular cells during the progression of diabetic vascular complications. In diabetic microangiopathy, hyperglycemia induces biochemical and molecular changes in microvascular cells that ultimately progress to retinal, renal, and neural complications and extends to other complications, including advanced periodontal disease. In this review, we describe changes involving basement membrane thickening, tissue remodeling, gap junctions, inflammation, cytokines, and transcription factors, and their effects on the pathogenesis of diabetic microvascular complications. The majority of the changes described relate to retinal microangiopathy, since ultrastructural, structural, and biochemical alterations have been well-characterized in this tissue.
    Journal of dental research 02/2010; 89(2):116-27. · 3.46 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Early and selective loss of pericytes and thickening of the basement membrane are hallmarks of diabetic retinopathy. We reported reduced adhesion, but no changes in apoptosis, of bovine retinal pericytes cultured on extracellular matrix (ECM) produced by endothelial cells in high glucose (HG). Since human and bovine pericytes may behave differently in conditions mimicking the diabetic milieu, we verified the behaviour of human retinal pericytes cultured on HG-conditioned ECM. Pericytes were cultured in physiological/HG on ECM produced by human umbilical vein endothelial cells in physiological/HG, alone or in the presence of thiamine and benfotiamine. Adhesion, proliferation, apoptosis, p53 and Bcl-2/Bax ratio (mRNA levels and protein concentrations) were measured in wild-type and immortalized human pericytes. Both types of pericytes adhered less to HG-conditioned ECM and plastic than to physiological glucose-conditioned ECM. DNA synthesis was impaired in pericytes cultured in HG on the three different surfaces but there were no differences in proliferation. DNA fragmentation and Bcl-2/Bax ratio were greatly enhanced by HG-conditioned ECM in pericytes kept in both physiological and HG. Addition of thiamine and benfotiamine to HG during ECM production completely prevented these damaging effects. Apoptosis is strongly increased in pericytes cultured on ECM produced by endothelium in HG, probably due to impairment of the Bcl-2/Bax ratio. Thiamine and benfotiamine completely revert this effect. This behaviour is therefore completely different from that of bovine pericytes, underlining the importance of establishing species-specific cell models to study the mechanisms of diabetic retinopathy.
    Diabetes/Metabolism Research and Reviews 09/2009; 25(7):647-56. · 2.97 Impact Factor

Full-text (2 Sources)

Available from
Jun 2, 2014