Calpains: a physiological regulator of the endothelial barrier?

Gonscherowski, Vera; Becker, Bernhard F.; Moröder, Luis; Motrescu, Elena; Gil-Parrado, Shirley; Gloe, Torsten; Keller, Matthias; Zahler, Stefan

doi:10.1152/ajpheart.00772.2004

Cited by 16 publications

(15 citation statements)

References 32 publications

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“…Immunofluorescence-HUVECs were isolated and cultivated as described previously (23). Briefly, cells were cultured on 8-chamber -slides (Ibidi, Munich, Germany).…”

Section: Methodsmentioning

confidence: 99%

RGD-dependent Binding of Procathepsin X to Integrin αvβ3 Mediates Cell-adhesive Properties

Lechner

Assfalg-Machleidt

Zahler

et al. 2006

Journal of Biological Chemistry

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Secreted lysosomal cysteine proteases (cathepsins) are involved in degradation and remodeling of the extracellular matrix, thus contributing to cell adhesion and migration. Among the eleven human lysosomal cysteine proteases, only procathepsin X contains an RGD motif located in a highly exposed region of the propeptide, which may allow binding of the proenzyme to RGD-recognizing integrins. Here, we have tested procathepsin X for cell-adhesive properties and found that it supports integrin ␣ v ␤ 3 -dependent attachment and spreading of human umbilical vein endothelial cells. Using sitedirected mutants of procathepsin X, we proved that this effect is mediated by the RGD sequence within the proregion of the protease. Endogenous procathepsin X is transported to the plasma membrane, accumulates in vesicles at lamellipodia of the human umbilical vein endothelial cell, and is partly associated with the cell surface, as shown by immunofluorescence. In addition, procathepsin X is partly co-localized with integrin ␤ 3 , as detected by immunogold electron microscopy. A direct interaction between endogenous procathepsin X and ␣ v ␤ 3 was demonstrated by co-immunoprecipitation. Moreover, surface plasmon resonance analysis revealed significant and RGD-dependent binding of procathepsin X to integrin ␣ v ␤ 3 . Our results provide for the first time evidence that the extracellular function of cathepsin X may include binding to integrins thereby modulating the attachment of migrating cells to ECM components. Cellular migration through extracellular matrix (ECM)2 requires both cell adhesion to and proteolysis of ECM components. A large number of proteases, including matrix metalloproteases, serine proteases, but also lysosomal cysteine proteases are known to be responsible for ECM breakdown, particularly during tumor invasion (1-3). Cell adhesion to ECM on the other hand is largely mediated by heterodimeric cell surface receptors of the integrin family. Integrins are composed of noncovalently associated ␣ and ␤ chains that form various heterodimers with distinct adhesive specificities (4, 5).Several proteases have been shown to mediate adhesion and migration of cells through interaction with integrins. Thus, prothrombin, the zymogen of a multifunctional serine protease of the blood coagulation system, interacts directly with integrins via its RGD sequence, a motif involved in integrin binding (6 -8). Although the RGD sequence is partly buried in the native conformation (9), prothrombin can bind in an RGD-dependent fashion to integrins ␣ IIb ␤ 3 of platelets and ␣ v ␤ 3 of cultured human endothelial cells (10 -12). There is increasing evidence that interactions between prothrombin and ␤ 3 integrins play important roles in the regulation of hemostatic and vascular functions. Furthermore, the urokinase-type plasminogen activator/urokinase receptor (uPA/ uPAR) system not only plays a key role in fibrinolysis but also regulates cell adhesion, cell migration, and cell proliferation through interaction with integrins (for review, see Ref...

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“…Immunofluorescence-HUVECs were isolated and cultivated as described previously (23). Briefly, cells were cultured on 8-chamber -slides (Ibidi, Munich, Germany).…”

Section: Methodsmentioning

confidence: 99%

RGD-dependent Binding of Procathepsin X to Integrin αvβ3 Mediates Cell-adhesive Properties

Lechner

Assfalg-Machleidt

Zahler

et al. 2006

Journal of Biological Chemistry

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“…Other studies have reported that inhibition of calpain activity improves organ function in diabetic animal models (11,12) and in acute cardiovascular events (13). In vitro studies have now implicated calpains in the regulation of endothelial cell barrier (14,15).…”

mentioning

confidence: 99%

Hyperglycemia Is a Major Determinant of Albumin Permeability in Diabetic Microcirculation

et al. 2007

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Increased permeability to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor of vascular complications. The mechanisms responsible for loss of the physiological albumin barrier in diabetic organs remain only partially understood. We have recently demonstrated that the protease -calpain is activated in hyperglycemia, which causes endothelial dysfunction and vascular inflammation. In the present study, we investigated whether -calpain is involved in the hyperpermeability of the diabetic vasculature. We also investigated the mechanistic roles of hyperglycemia and leukocyte adhesion in this process. Albumin permeability in the intact microcirculation of the Zucker diabetic fatty (ZDF) rat was quantified by intravital microscopy. Extravasation of albumin in the microcirculation of ZDF rats was significantly increased when compared with nondiabetic Zucker lean (ZL) rats. Microvascular albumin leakage was prevented by either antisense depletion of -calpain or pharmacological inhibition of calpain in vivo. Calpain inhibition also attenuated urinary albumin excretion in ZDF rats. Glucose concentrations in the range of those found in the blood of ZDF rats increased albumin permeability in nondiabetic ZL rats. Thus, this demonstrates a mechanistic role for hyperglycemia in the hypermeability of diabetes. Depletion of polymorphonuclear leukocytes in vivo failed to prevent glucose-induced hypermeability, which suggests that hyperglycemia can disrupt the physiological endothelial cell barrier of the microcirculation, even in the absence of increased overt leukocyte-endothelium interactions.

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“…Mechanistically, calpainmediated regulation of small GTPases 13,40) as well as calpain-induced proteolysis of focal adhesion proteins 37) during EC dynamics has been documented previously. Accordingly, calpain systems appear to play key roles in angiogenesis 36,38) , wound closure 39) and maintenance of barrier functions 13,40) in ECs. In addition to motility responses, it has been reported that nitric oxide (NO) production in ECs is modulated through calpain-induced proteolysis of HSP90 42) or calpain-regulated PI3K/AMPK signaling 35) .…”

Section: Calpain and Vascular Integritymentioning

confidence: 73%

“…It is likely that VEGF 35,36) and fluid shear stress 13,37,38) are representative activators of calpain systems in ECs under physiological conditions. ECs exhibit measurable calpain activity even under unstimulated conditions 39,40) , which may be due to spontaneous Ca 2＋ mobilization in the cells 41) . Mechanistically, calpainmediated regulation of small GTPases 13,40) as well as calpain-induced proteolysis of focal adhesion proteins 37) during EC dynamics has been documented previously.…”

Section: Calpain and Vascular Integritymentioning

confidence: 99%

“…ECs exhibit measurable calpain activity even under unstimulated conditions 39,40) , which may be due to spontaneous Ca 2＋ mobilization in the cells 41) . Mechanistically, calpainmediated regulation of small GTPases 13,40) as well as calpain-induced proteolysis of focal adhesion proteins 37) during EC dynamics has been documented previously. Accordingly, calpain systems appear to play key roles in angiogenesis 36,38) , wound closure 39) and maintenance of barrier functions 13,40) in ECs.…”

Section: Calpain and Vascular Integritymentioning

confidence: 99%

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