Autoestima alta

The in vitro properties of fibroblasts and endothelial cells strongly suggest that functional interactions, or cross talk, between these cells in vivo might be important. Such interactions could be mediated directly by soluble products of each cell type acting on the other, or indirectly though their effect on extracellular matrix, cell shape or through recruitment of other cellular elements such as platelets or leucocytes.

Table 1.7 Scleroderma fibroblast phenotype in vitro

property findings

36

references

Extracellular matrix turnover Collagen synthesis Fibronectin synthesis Proteoglycan synthesis Glycolipid secretion Collagenase TIM? levels a-actin expression

Adhesion molecule expression ICAM-1

ectopeptidase expression (31 integrins

Cytokine release and responsiveness IL-1 IL-6 PDGF TGFP ET-I oncostatin calmodulin

increased types 1, III and VI protein

elevated steady state mRNA for type I and III collagen transcriptional activation C0LIA2 and COLIAI elevated collagen pro-peptide secretion

impaired collagen (I) mRNA down-regulation in 3-D collagen gel increased

increased increased

biological activity reduced TIMPI increased

increased

increased surface expression increased up-regulation by cytokines greater shedding of sICAM-1 increased

aberrant expression of collagen binding integrins greater functional responses (ICAM-1 expression

and proliferation) increased expression receptor and ligand increased secretion and mRNA levels

increased secretion and receptor upregulation by TGPp increased proliferative response

induces PDGF receptor upregulation

increased secretion, reduced mitogenic response less induction of extracellular matrix synthesis increased intracellular levels

LeRoy, 1974; Graves et al, 1983

Kikuchi et al, 1992; Hitreya and Jimenez, 1996 Kikuchi et ûf/, 1995

Ivarsson et al, 1993; Eckes et al, 1996 Eckes et ûf/, 1996

Komleva et al, 1995; Westergren-Thorson et al, 1996 Komleva et a/, 1995

Takeda et a/, 1994

Kirk et al, 1995; Bou-Gharios et al, 1994

Abraham et al, I99I Cho et al, 1994 Shi-wen et ûf/, 1994 Bou-Gharios et al, 1995

Ivarsson et al, 1993; Kozlowska et al, 1996

Kawaguchi et al, 1992; 1994 Feghali et al, 1994

Yamakage et al, 1992 Kikuchi et al, 1995a Yamakage et al, 1992

Kawaguchi et al, 1994; Kikuhci et al, 1995b Duncan, et al, 1995

Table 1.8 Classification of the metalloproteinases

37

Enzyme Abbreviation MMP# Mr Extracellular matrix substrates

Fibroblast-type collagenase

FIB-CL MMP-1 57000/

52000

collagen I, II, III (III>I), VII, VIII, X gelatin; PG core protein

PMN-type collagenase

PMN-CL MMP-8 75000 same as FIB-CL (I>III)

Stromelysin-1 SL-1 MMP-3 60000

55000

PG core protein; fibronectin; laminin collagen IV, V, IX, X, elastin; proCL

Stromelysin-2 SL-2 MMP-10 60000/ 55000 same as SL-1 Stromelysin-3 SL-3 MMP-11 n.d. n.d. Macrophage metallo-elastase MME ? 53000 elastin Mr 72K gelatinase type IV collgenase

Mr 72K GL MMP-2 72000 gelatin; collagen IV, V, VII, X, XI; elastin

fibronectin; PG core protein

Mr 92K gelatinase type IV collgenase

Mr92K GL MMP-9 92000 gelatin; collagen IV, V; elastin; PG core protein

Putative metallo­ proteinase-1

PUMP-1 MMP-7 28000 fibronectin, laminin, collagen IV, gelatin, proCL

PG core protein

The feasibility o f such an interplay is supported by the close juxtaposition o f endothelial cells and fibroblasts in most tissues, and by the range of soluble products of each cell type which might influence the other (Kahaleh, 1992). Some specific examples of pathological circumstances when altered endothelial-fibroblast cross talk may occur and the possible mechanisms involved are discussed below.

1.4.1 Molecular mechanisms for intercellular cross-talk

Endothelial cells have been reported to produce a large number of cytokines and growth factors. These include IL-1 (Cozzolini et al. 1990), IL-6 (Sironi et al. 1989), bFGF (Brooks

et a l 1991), ET-1 (Wagner et a l 1992), EL-8, MCP-1, IGF-1, GM-CSF (Swerlick and Lawley, 1993), TGF-p (Antonelli-Orlidge et a l 1989) and CTGF (Bradham et a l 1991). Other cytokine products have also been suggested (e.g. PDGF, TNFa) but studies have produced conflicting results. All of these factors have been shown to modulate fibroblast properties (see Table 1.6) and so would be candidate mediators for endothelial cell -induced modulation of fibroblast properties. Most exert complex effects on fibroblasts and the precise effects will depend on other factors such as fibroblast-matrix interaction and cell shape as discussed above. The potent mitogenic effect of endothelial cell-conditioned medium is believed to arise through a combination of endothelial cell-derived mitogenic factors (McNeil

et a l 1989; Eguchi et a l 1992). Certainly bFGF, ET-1 and CTGF have been demonstrated to have this effect and other factors appear to have a more specific effect on protein metabolism (Villanueva et a l 1991). It is possible that feedback inhibitory signals from connective tissue cells, such as fibroblasts or smooth muscle cells, act on endothelial cells to reduce the secretion of activating factors. It has been shown that ET-1 secretion is inhibited in co­ culture with mesenchymal cells (Stewart et a l 1990).

Fibroblast products are also diverse and their production and release is modulated by extrinsic influences. However studies of mRNA levels by northern hybridisation, in situ hybridisation and RT-PCR suggest that several relevant factors may be synthesised including IL-1, IL-6, IGF-1 (Eckes et a l 1992; Kupper and Groves, 1995; Weber et a l 1995). It is less clear to what extent these mRNA transcripts are translated, and moreover, whether any proteins synthesised are released in an active form so that they can modulate endothelial cell properties. Theoretically, however, the case is strong since these factors are known to

modulate endothelial cell properties. Other studies have obtained direct evidence for fimctional interactions between endothelial cells and fibroblasts (Eguchi et al. 1992; Guarda et al. 1993).

1.4.2 Wound healing

One circumstance in which fibroblasts and endothelial cells are likely to interact is in the process of wound healing, which represents a non-specific response to tissue injury, and is characterised by the formation of vascular connective tissue in its early stages (Falanga et al.

1996). Neo-vascularisation and scar formation with fibrosis proceed in a synchronous and apparently coordinated fashion, suggesting that there is regulated growth and differentiation o f vascular and mesenchymal cells. One mechanism for this, which is supported by tissue culture models, is through the paracrine effects of soluble mediators produced by one or both cell types. These interactions were recently reviewed by Swerlick and Lawley, (1993). There are analogies between the fibrosis occurring in scleroderma and that seen in scar formation, especially in situations of pathological healing which result in keloid or hypertrophic scar formation. It is interesting therefore that studies of the cytokine responsiveness of hypertrophic scar fibroblasts have identified abnormal patterns of response, including to factors which may be released by endothelial cells (Gamer et al. 1993).

1.4.3 Post angioplasty re-stenosis

Another situation in which a close relationship exists between endothelial cells and fibroblasts is in the situation of vascular stenosis after angioplasty (Brady and Warren, 1991). This is an important clinical problem, and one into which there has been a large amount of experimental investigation, examining the responses of endothelial cells and other vascular components to injury (Madri et al. 1991) It is suggested that endothelial cell-derived growth factors both released from the cells and mobilised from the sub-endothelial cell basement membrane are responsible for initiating fibroblast and smooth muscle cell proliferation which leads to a fibrotic stenosis at the site of angioplasty (Vlodavsky et al. 1991). Studies have tried to modulate the effect of endothelial cell derived factors for example using heparin or neutralising antibodies or novel molecular strategies to try to reduce the severity of stenosis (McEwan a/. 1994).

1.4.4 Atherogenesis

Atherosclerosis and its complications are a major cause for morbidity and mortality in many populations, and coronary artery disease is the single most frequent cause of death in Western Europe and the USA. The histology of advanced atheroma includes fibrosis as well as endothelial damage, which suggests functional links between endothelial cell and fibroblasts in the pathogenesis of atherosclerosis (Ross, 1993). Similar mechanisms to those discussed above could induce fibro-proliferative lesions following endothelial cell perturbation. However many other cell types have also been implicated in the development of atheroma and endothelial cell-fibroblast interactions are hkely to reflect only one aspect on the pathogenesis of this important condition.