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...h VN, but rather due to prevention of excessive plasmin formation. Discussion Proteolytic breakdown of extracellular matrices by uPA/ plasmin has been associated with tumor invasion and angiogenesis (=-=Andreasen et al., 1997-=-; Stephens et al., 1999). However, prognostic studies have indicated that the protease inhibitor PAI-1 is a clinical marker of poor prognosis in a variety of human cancers (Pedersen et al., 1994a,b; B...

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...osis of the trimolecular uPA–PAI-1–uPAR complex (Conese and Blasi, 1995; Blasi, 1997). However, it has also been implicated in modulating cell migration via alternative mechanisms (Deng et al., 1996; =-=Stefansson and Lawrence, 1996-=-; Blasi, 1997; Loskutoff et al., 1999), even though conclusive in vivo evidence is lacking. By blocking the interaction between vitronectin (VN), uPAR, and integrins, PAI-1 may induce cell detachment ...

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...None 9 ND �� �� None 5 ND ��� ��� Plg��� None 5 ND � 0 None 3 ND � � Tumor invasion and angiogenesis were scored as described in Materials and Methods. found to promote tumor growth and angiogenesis (=-=Bajou et al., 1998-=-) in a highly reproducible mouse tumor model. The availability of this model now allows us to examine the molecular mechanism by which PAI-1 promotes tumor angiogenesis (i.e., either by inhibiting the...

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...al conditions can occur to a large degree in the absence of either Plg, tPA, or uPA, possibly by redundancy or compensation by other proteinases (Bugge et al., 1997, 1998; Carmeliet and Collen, 1999; =-=Lund et al., 1999-=-). In accordance with these observations, the present findings show that tumor vascularization was not affected in mice deficient in tPA, uPA, or uPAR. Notably, stromal/host lack of uPA may in the pre...

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...ated PAI-1 cDNA Transfer A recombinant adenovirus vector bearing WT human PAI-1 (hPAI-1) (AdCMVPAI-1 and control adenovirus [AdRR5]) were propagated as described previously (Gerard and Meidell, 1995; =-=Carmeliet et al., 1997-=-). Recombinant viruses expressing mutant hPAI-1, AdCMVPAI-1 Q123K , and AdCMVPAI-1 R346M,M347S were generated after substitution of a restriction fragment containing the desired mutation into the pACC...

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...g the rapid endocytosis of the trimolecular uPA–PAI-1–uPAR complex (Conese and Blasi, 1995; Blasi, 1997). However, it has also been implicated in modulating cell migration via alternative mechanisms (=-=Deng et al., 1996-=-; Stefansson and Lawrence, 1996; Blasi, 1997; Loskutoff et al., 1999), even though conclusive in vivo evidence is lacking. By blocking the interaction between vitronectin (VN), uPAR, and integrins, PA...

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...activity. First, PAI-1 is known to interact with VN and, consequently, has been considered as a molecular switch governing uPAR- and/or integrinmediated cell adhesion and motility (Deng et al., 1996; =-=Chapman, 1997-=-). Binding of PAI-1 to VN blocks the interaction between integrins and the uPAR–uPA complex with VN, thereby inhibiting cell adhesion and migration (Loskutoff et al., 1999). However, in our transplant...

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...giogenesis during pathological conditions can occur to a large degree in the absence of either Plg, tPA, or uPA, possibly by redundancy or compensation by other proteinases (Bugge et al., 1997, 1998; =-=Carmeliet and Collen, 1999-=-; Lund et al., 1999). In accordance with these observations, the present findings show that tumor vascularization was not affected in mice deficient in tPA, uPA, or uPAR. Notably, stromal/host lack of...

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...mediates its proangiogenic effect, not by its interaction with VN but rather by its antiprotease function. The Plg/plasmin system has been implicated in extracellular proteolysis during angiogenesis (=-=Montesano et al., 1990-=-). However, studies in gene-inactivated mice have revealed that angiogenesis during embryonic development occurs normally in the absence of both PAs and Plg and that angiogenesis during pathological c...

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... with C57BL/6 strain. In each set of experiments, similar results were obtained and therefore the data presented in Table I were pooled. The Plg ��� mice were derived from those generated previously (=-=Bugge et al., 1995-=-; Ploplis et al., 1998). They have been backcrossed 1 or 11 times with C57BL/6 strain, yielding similar results in two different sets of experiments presented in Table I. Mice of either sex used for e...

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...deficiencies (tPA ��� /uPA ��� mice) and their corresponding WT with a mixed genetic background of 75% C57BL/6 and 25% 129 SV/SL strain were generated as described previously (Carmeliet et al., 1994; =-=Zheng et al., 1995-=-; Carmeliet and Collen, 1998). Tumor angiogenesis and invasion in PAI-1 ��� (Carmeliet et al., 1993a,b) and WT mice were evaluated in three different sets of experiments using mice with different gene...

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...tent/full/152/4/777 777addition, the use of antisense mRNA for uPA and uPAR, of natural or synthetic serine protease inhibitors, or of uPAR antagonists, all reduced tumor invasion (Min et al., 1996; =-=Carmeliet and Collen, 1998-=-). Consequently, uPA/ uPAR/plasmin antagonists are currently being developed as therapeutic strategies to inhibit tumor angiogenesis and progression. PA inhibitor 1 (PAI-1) is the primary physiologica...

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...ld allow newly formed vessels to acquire the necessary stability and maturity. Excessive degradation of extracellular matrix is incompatible with efficient cellular migration (Montesano et al., 1990; =-=Pepper and Montesano, 1990-=-). The maintenance of a certain degree of extracellular matrix integrity is indeed an essential requirement for capillary morphogenesis. PAI-1 may thus balance PA-mediated pericellular proteolysis, pr...

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...is conclusion is supported by the fact that uPA and uPAR are highly expressed by tumor cells or by surrounding stromal cells, and that they are both independent prognostic indicators in human cancer (=-=Dano et al., 1994-=-; Reuning et al., 1998; Stephens et al., 1999). In © The Rockefeller University Press, 0021-9525/2001/02/777/8 $5.00 The Journal of Cell Biology, Volume 152, Number 4, February 19, 2001 777–784 http:/...

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... angiogenesis has not been confirmed in vivo. Surprisingly high, rather than low, levels of PAI-1 are predictive of poor survival prognosis for patients suffering from a variety of different cancers (=-=Pedersen et al., 1994-=-a,b). To date, the molecular mechanisms of this apparent paradox remain largely unexplained, raising concerns about whether therapeutic strategies to suppress tumor growth and angiogenesis should be a...

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...pported by the fact that uPA and uPAR are highly expressed by tumor cells or by surrounding stromal cells, and that they are both independent prognostic indicators in human cancer (Dano et al., 1994; =-=Reuning et al., 1998-=-; Stephens et al., 1999). In © The Rockefeller University Press, 0021-9525/2001/02/777/8 $5.00 The Journal of Cell Biology, Volume 152, Number 4, February 19, 2001 777–784 http://www.jcb.org/cgi/conte...

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... angiogenesis has not been confirmed in vivo. Surprisingly high, rather than low, levels of PAI-1 are predictive of poor survival prognosis for patients suffering from a variety of different cancers (=-=Pedersen et al., 1994-=-a,b). To date, the molecular mechanisms of this apparent paradox remain largely unexplained, raising concerns about whether therapeutic strategies to suppress tumor growth and angiogenesis should be a...

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.... In each set of experiments, similar results were obtained and therefore the data presented in Table I were pooled. The Plg ��� mice were derived from those generated previously (Bugge et al., 1995; =-=Ploplis et al., 1998-=-). They have been backcrossed 1 or 11 times with C57BL/6 strain, yielding similar results in two different sets of experiments presented in Table I. Mice of either sex used for experiments were litter...

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...th PAs and Plg and that angiogenesis during pathological conditions can occur to a large degree in the absence of either Plg, tPA, or uPA, possibly by redundancy or compensation by other proteinases (=-=Bugge et al., 1997-=-, 1998; Carmeliet and Collen, 1999; Lund et al., 1999). In accordance with these observations, the present findings show that tumor vascularization was not affected in mice deficient in tPA, uPA, or u...

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...teolysis, protecting the stroma from excessive proteolysis during endothelial cell invasion. In conclusion, our data indicate that in vivo, like previously shown in vitro (Pepper and Montesano, 1990; =-=Liu et al., 1995-=-), a critical balance between proteases and PAI-1 is necessary for optimal invasion. However, in contrast to in vitro data, lack of VN, uPAR, uPA, tPA, or both uPA and tPA did not impair tumor formati...

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...f uPA and tPA. It not only regulates the proteolytic activity of uPA, but also determines the level of uPA bound to uPAR by promoting the rapid endocytosis of the trimolecular uPA–PAI-1–uPAR complex (=-=Conese and Blasi, 1995-=-; Blasi, 1997). However, it has also been implicated in modulating cell migration via alternative mechanisms (Deng et al., 1996; Stefansson and Lawrence, 1996; Blasi, 1997; Loskutoff et al., 1999), ev...

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... injected. Adenovirus-mediated PAI-1 cDNA Transfer A recombinant adenovirus vector bearing WT human PAI-1 (hPAI-1) (AdCMVPAI-1 and control adenovirus [AdRR5]) were propagated as described previously (=-=Gerard and Meidell, 1995-=-; Carmeliet et al., 1997). Recombinant viruses expressing mutant hPAI-1, AdCMVPAI-1 Q123K , and AdCMVPAI-1 R346M,M347S were generated after substitution of a restriction fragment containing the desire...

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...agen gel (4 mg/ml of type I collagen isolated from rat tail tendons) inserted in teflon rings (Renner GmbH) and maintained in culture for 1 d before transplantation into mice as described previously (=-=Fusenig et al., 1983-=-; Bajou et al., 1998). For transplantation assays in VN ��� mice, keratinocytes were precultured on the collagen gels in the presence of 10% serum derived from VN ��� mice. Transplantation Assay in Mi...

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...at uPA and uPAR are highly expressed by tumor cells or by surrounding stromal cells, and that they are both independent prognostic indicators in human cancer (Dano et al., 1994; Reuning et al., 1998; =-=Stephens et al., 1999-=-). In © The Rockefeller University Press, 0021-9525/2001/02/777/8 $5.00 The Journal of Cell Biology, Volume 152, Number 4, February 19, 2001 777–784 http://www.jcb.org/cgi/content/full/152/4/777 777a...

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...ablished using tail biopsy DNA preparation by either Southern blot analysis or PCR assays (Carmeliet et al., 1993b, 1994; Bugge et al., 1995). Cell Culture Malignant murine keratinocytes (PDVA cells; =-=Fusenig et al., 1978-=-) were routinely grown in DME containing a fourfold concentration of amino acids and vitamins (GIBCO BRL), 10% FCS (GIBCO BRL), and antibiotics in a humidified incubator at 37�C, 5% CO 2. Cells (2 � 1...

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...lex (Conese and Blasi, 1995; Blasi, 1997). However, it has also been implicated in modulating cell migration via alternative mechanisms (Deng et al., 1996; Stefansson and Lawrence, 1996; Blasi, 1997; =-=Loskutoff et al., 1999-=-), even though conclusive in vivo evidence is lacking. By blocking the interaction between vitronectin (VN), uPAR, and integrins, PAI-1 may induce cell detachment from the extracellular matrix and the...

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...r cells (Bajou et al., 1998). The Plg activation pathway(s) occurring in the double-deficient mice remain(s) to be determined, but might involve blood coagulation factor XII, kininogen, or kalikrein (=-=Colman, 1969-=-; Miles et al., 1983; Carmeliet et al., 1994). The reduced tumor vascularization and invasion in Plg ��� mice indicate that plasmin is required for optimal tumor progression. The fact that some tumor ...

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... et al., 1998). The Plg activation pathway(s) occurring in the double-deficient mice remain(s) to be determined, but might involve blood coagulation factor XII, kininogen, or kalikrein (Colman, 1969; =-=Miles et al., 1983-=-; Carmeliet et al., 1994). The reduced tumor vascularization and invasion in Plg ��� mice indicate that plasmin is required for optimal tumor progression. The fact that some tumor angiogenesis was obs...

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... affinity as WT PAI-1 but did not inhibit PA activity. We verified that hPAI-1 (WT or mutant forms) interacts efficiently with murine PA and murine VN according to the procedure described previously (=-=Vleugels et al., 2000-=-). 1 d after cell transplantation, mice were intravenously injected with 200 �l of control or recombinant adenovirus (7 � 10 8 pfu). After 5 d, blood was sampled from the retroorbital sinus and PAI-1 ...

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... Gln 123 to Lys had a specific 100-fold decrease in affinity for VN, but retained full inhibitory activity (Stefansson and Lawrence, 1996). The double point mutant, Arg 346 to Met and Met 347 to Ser (=-=Shubeita et al., 1990-=-), bound to VN with the same affinity as WT PAI-1 but did not inhibit PA activity. We verified that hPAI-1 (WT or mutant forms) interacts efficiently with murine PA and murine VN according to the proc...