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Sympathetic postganglionic neuronal involvement in tissue-type plasminogen activator release
24
FIBRINOLYSIS
Taken together, thesedata suggest a role for PA/PA1 in periodontal pathophysiology, most intriguinglyin thediierentiation program ofthis unique type of stratified sqUarn0US
76
eplthelltmn
SECRETION OF TISSUE-TYPE PLASMINOGEN ACTlVATOR AND PLA9MlNOGEN ACTNATOR INHIBITOR-1 BY CULTURED HUMAN RETINAL ENDOTHEUAL CELLS: MODULATlON BY INSULIN-UKE GROWTH FACTOR 1 Maria 6. Grant and C&an Gwy fkparfmant of Me&he, UnivtMy
ofFbrick, GainasviIa, Florida, USA
Growth hormone (GH), which is postulated to influence the development of diabetic retinopathy, has its principle actions mediated by insulin-like growth factor 1 (IGF). Elevated IGF 1 serum 78 concentrations are associated with a rapid acceleration of proliferative retinopathy, and IGF 1 concentrations in vitreous of diabetics are greater than in controls. IGF 1 has also been shown to have a chemotactic efiect on human retinal endothelial cells (HREC). The currentstudyconsiderstheinfluenceofIGF landselectedothergrowth factors onthe synthesis oftissuetype plasminogen activator (t-PA) and its natural inhibitor (PAI-1) by HREC frcm ncndiabetic and diabetic subjects. CPA converts plasminogen to plasmin, which can cause proteolysis of many extracellular matrix components includinglaminin and fibtcnectin. t-PA, therefore, may have a role in degradation of basement membranes - a key step in neovasculartxaticn. Three cell lines derived from nondiabetic indiviiuals and three cell diabetic individualswere tested using ELlSAfor t-PA lines derived antigen and PAI- antigen. Results are expressed in @ml and representthe mean& SEM of tripliie &u&s. Growth factors studled were IGF 1, epidermal growth factor (EGF) and acidic fibroblastic growthfactor (AFGF).
from
t-PA r.&nr&b& L&b&
PAI hbndiabelic
LXabe&z
Contrd lO.lM.8 9.5io.6 6.1M.i 6til5 IGF1(lO@$mi) 10.7fl.l 16.6ct1.9 pdulO1 6.8ztl.O 11kt3.4 EGF(16fJqtnl) 6.4ztz.O 14.6t2.7 3.&63 pcR.661 12.9ztZ.6 AFGF(lOLk@ml) 9.6ho.7 6.(lto.4 3.983.4 pdl.rl6s 7.1f1.6
Condusion: HREC of &&tic origin respond to IGF 1 with a marked increase of t-PA production compared to unstimulated cells; HREC of nondabetic origin do not. IGF I does not affect PAI-I production of HREC of &her nondiabetic or diabetic origin. AFGF and EGF actually depress production of PAl-1, but only in HREC of nondiabetic orlgln. 77
SYMPATHETIC POSTGANGUONIC NEURONAL INVOLVEMENT IN TISSUE-TYPE PLASMINOGEN ACTlVATOR RELEASE M. Grtw$ C. Guayand R. Loftenberg Dqartmant
of Mcxkina, University of FkMa,
Gainastilie, Florida, USA
The mechanism controlling the release of tissue type plasminogen activator(t-PA) from endothelial cells is unknown. We have shownthat the release of t-PA following desmopressin acetate (DDAVP) infusion was associated with a selective and parallel rise of plasma norepinephrine (NE). The absence of a detectable change in plasma epinephrine provided strong evidence that the plasma norepinephrine response to DDAVP stimulation was derived predominantly if not exdusively from sympathetic postganglibnic neurons (Thromb and Haemosta 59:289272,1088). It was then hypoIhesized that DDAVP mayamplifyendogenowchdinergicactivity,possiblythroughacentral mechanism, resulting in a drect stimulation of sympathetic post gangliinic neurons. At the plasma levels of norepinephrlne achieved following DDAVP, the neumtransmllter norepinephrlne may serve a hormonal function acting on embthelial cells and possibly stimulating t-PA release. Further stucliesexamining this hypothesis used Tensibn to amplify endogenous chdinergic activity and directly sfimulate sympathetic postganglicnic neurons. Twenty subjects were fasted for 12 hours prior to beginning the study and were maintained at strict bed rest for a minimum oftwo hours prior to testing. Plasma was collected at the slpine state at 2.5 min intervals for 80 minutes following the administration of IO mg Tensibn IV push. Measurements of t-PA antigen, plasminogen activator inhibitor antigen, von Willebrand factor, and fractionated catecholamines were obtained. NE levels rose in all s&+zcts. Basal levels were 184f20 pg/ml and increased to 28Oi32 pg/ml, Pd3.0005,
basal t-PA was 5.5BtI.85 ng/ml and rose to iO.g2i2.84@‘d, Pd).0015,basalPAIwas5.07~.05nglmlandinaeesedto10.~.~ ng/ml, PcO.005 and basal vWF was lOQt28% and increased to I_%, pcD.0008. In selected patients, the sympathetic nervous system was stimulatedby changing the sub@& position, from supine to standing. A six-fold increase in t-PA levels over basal levels md a three-fold increase in NE levels over basal levels was demon&r&d. In surnmq, augmented norepinephtfne release, eitherthrough a central mechanism such as with DDAVP, or thrcugh a peripheral mechanism with Tensibn results in release of tissue plasmktogan activator. Our data support a role for NE in rnc&lating the release of key proteins involved in coagulation and fibrlnoiysis.
MUSCLE PLASMINOGEN ACTlVATlON IN THE WOBBLER MUTANT MOUSE Dankltlant9~ Geo#3k%flovafz4fe/mon, &i#fteBlon~ Fhncois R/eger, Jeannette So& Ckwdlne SoHa andBarry W. Fhstoff (I) Paris, Francs and (l)Kansas C@, MO, USA The neurological mutant mouse Wobbler carries an autosomal recessive gene (wr) and has been studied as a model of lower motor neuron disorders with associated muscle atrophy and dsnervatbnlreinnervati~ phenomena (1). In ad&ion a recent report has identified neuromuscular junction defects and, hence, it may also be a modal for defects of synapse formation (2). In previous studies we have shown that during mouse neuromuscular development a decrease in muscle PA activities accompanies synapse maturation (3). Experimental denewation in aduHmiceleedsontheotherhandtoanimxeaseinmusdePAectivities (4). These musde PAS might then ba responsble for the degradstion of scme components of the muscle basement membrane (5), thereby setting the stage for reinnervation after denervation (6). In order to detem-rine the possible involvement of PAS in the basement membrane degradation associated with denervation observedinWcbMer,specificPAac6vitiesinbicepsandgasImcnemius musdeexbactswereestimatedusingbdhzymographyandasynthetic &&rate (B2251, K&i) relatively spsciticforplasmin. These activities were compared to those of the corresponding muscles in unaffeded mice (wr/+) from the same litter. PA activity was increased in Wcbbler mousemusclesversuscontrolmusdesandmoreinbicepsmusdethan in gastromemius musde, which is less inpaired by the mutation. Muscle PA was mostly uPA (f&=48 kD) more Ihan tPA (Mt=75 kD). In addiion, kinetlccharacteristics ofthe PA assay obtained with Wobbler mouse muscles lackedtheccnventbnal lag-phase,thus evidencing the presence of already formed plasmin. The neurological mutant mouse Wobbler is an autosomal recessive disorder that approximates the human lower motor neuron diseases (1) such as the fatal Amyotrophic Lateral Bderosis (ALS). The present results support the hypothesis of an activation of specific serine proteases in the pathogenesis of ALB (7). In additton, study of Wobbler PA regulaticn in muscle may shed light on the role of these cascades, and their inhibitors, in neuromuscular fomdon. 1. Andrewset al., Am. J. Path. 76,63-76. 1974. 2. La Vail and Irons, Brain Res. 463,78-89, 1988. 3. Hantai et al., Pm. Nati. Acad. Sci. USA, 86,38X366, 1989 4. Festofietal., J. Cell Biol. 193, 14161421, 1986. 5. Hantai and Festoff,Exp. Neural. 95,44-55.1987. 6. Hantai etal., Rev. Neural. (Paris) 144,660-667,1988. 7. Festoff,Med. Hypotheses6,121-131,198O.
FIBRINOLYSIS
Taken together, thesedata suggest a role for PA/PA1 in periodontal pathophysiology, most intriguinglyin thediierentiation program ofthis unique type of stratified sqUarn0US
76
eplthelltmn
SECRETION OF TISSUE-TYPE PLASMINOGEN ACTlVATOR AND PLA9MlNOGEN ACTNATOR INHIBITOR-1 BY CULTURED HUMAN RETINAL ENDOTHEUAL CELLS: MODULATlON BY INSULIN-UKE GROWTH FACTOR 1 Maria 6. Grant and C&an Gwy fkparfmant of Me&he, UnivtMy
ofFbrick, GainasviIa, Florida, USA
Growth hormone (GH), which is postulated to influence the development of diabetic retinopathy, has its principle actions mediated by insulin-like growth factor 1 (IGF). Elevated IGF 1 serum 78 concentrations are associated with a rapid acceleration of proliferative retinopathy, and IGF 1 concentrations in vitreous of diabetics are greater than in controls. IGF 1 has also been shown to have a chemotactic efiect on human retinal endothelial cells (HREC). The currentstudyconsiderstheinfluenceofIGF landselectedothergrowth factors onthe synthesis oftissuetype plasminogen activator (t-PA) and its natural inhibitor (PAI-1) by HREC frcm ncndiabetic and diabetic subjects. CPA converts plasminogen to plasmin, which can cause proteolysis of many extracellular matrix components includinglaminin and fibtcnectin. t-PA, therefore, may have a role in degradation of basement membranes - a key step in neovasculartxaticn. Three cell lines derived from nondiabetic indiviiuals and three cell diabetic individualswere tested using ELlSAfor t-PA lines derived antigen and PAI- antigen. Results are expressed in @ml and representthe mean& SEM of tripliie &u&s. Growth factors studled were IGF 1, epidermal growth factor (EGF) and acidic fibroblastic growthfactor (AFGF).
from
t-PA r.&nr&b& L&b&
PAI hbndiabelic
LXabe&z
Contrd lO.lM.8 9.5io.6 6.1M.i 6til5 IGF1(lO@$mi) 10.7fl.l 16.6ct1.9 pdulO1 6.8ztl.O 11kt3.4 EGF(16fJqtnl) 6.4ztz.O 14.6t2.7 3.&63 pcR.661 12.9ztZ.6 AFGF(lOLk@ml) 9.6ho.7 6.(lto.4 3.983.4 pdl.rl6s 7.1f1.6
Condusion: HREC of &&tic origin respond to IGF 1 with a marked increase of t-PA production compared to unstimulated cells; HREC of nondabetic origin do not. IGF I does not affect PAI-I production of HREC of &her nondiabetic or diabetic origin. AFGF and EGF actually depress production of PAl-1, but only in HREC of nondiabetic orlgln. 77
SYMPATHETIC POSTGANGUONIC NEURONAL INVOLVEMENT IN TISSUE-TYPE PLASMINOGEN ACTlVATOR RELEASE M. Grtw$ C. Guayand R. Loftenberg Dqartmant
of Mcxkina, University of FkMa,
Gainastilie, Florida, USA
The mechanism controlling the release of tissue type plasminogen activator(t-PA) from endothelial cells is unknown. We have shownthat the release of t-PA following desmopressin acetate (DDAVP) infusion was associated with a selective and parallel rise of plasma norepinephrine (NE). The absence of a detectable change in plasma epinephrine provided strong evidence that the plasma norepinephrine response to DDAVP stimulation was derived predominantly if not exdusively from sympathetic postganglibnic neurons (Thromb and Haemosta 59:289272,1088). It was then hypoIhesized that DDAVP mayamplifyendogenowchdinergicactivity,possiblythroughacentral mechanism, resulting in a drect stimulation of sympathetic post gangliinic neurons. At the plasma levels of norepinephrlne achieved following DDAVP, the neumtransmllter norepinephrlne may serve a hormonal function acting on embthelial cells and possibly stimulating t-PA release. Further stucliesexamining this hypothesis used Tensibn to amplify endogenous chdinergic activity and directly sfimulate sympathetic postganglicnic neurons. Twenty subjects were fasted for 12 hours prior to beginning the study and were maintained at strict bed rest for a minimum oftwo hours prior to testing. Plasma was collected at the slpine state at 2.5 min intervals for 80 minutes following the administration of IO mg Tensibn IV push. Measurements of t-PA antigen, plasminogen activator inhibitor antigen, von Willebrand factor, and fractionated catecholamines were obtained. NE levels rose in all s&+zcts. Basal levels were 184f20 pg/ml and increased to 28Oi32 pg/ml, Pd3.0005,
basal t-PA was 5.5BtI.85 ng/ml and rose to iO.g2i2.84@‘d, Pd).0015,basalPAIwas5.07~.05nglmlandinaeesedto10.~.~ ng/ml, PcO.005 and basal vWF was lOQt28% and increased to I_%, pcD.0008. In selected patients, the sympathetic nervous system was stimulatedby changing the sub@& position, from supine to standing. A six-fold increase in t-PA levels over basal levels md a three-fold increase in NE levels over basal levels was demon&r&d. In surnmq, augmented norepinephtfne release, eitherthrough a central mechanism such as with DDAVP, or thrcugh a peripheral mechanism with Tensibn results in release of tissue plasmktogan activator. Our data support a role for NE in rnc&lating the release of key proteins involved in coagulation and fibrlnoiysis.
MUSCLE PLASMINOGEN ACTlVATlON IN THE WOBBLER MUTANT MOUSE Dankltlant9~ Geo#3k%flovafz4fe/mon, &i#fteBlon~ Fhncois R/eger, Jeannette So& Ckwdlne SoHa andBarry W. Fhstoff (I) Paris, Francs and (l)Kansas C@, MO, USA The neurological mutant mouse Wobbler carries an autosomal recessive gene (wr) and has been studied as a model of lower motor neuron disorders with associated muscle atrophy and dsnervatbnlreinnervati~ phenomena (1). In ad&ion a recent report has identified neuromuscular junction defects and, hence, it may also be a modal for defects of synapse formation (2). In previous studies we have shown that during mouse neuromuscular development a decrease in muscle PA activities accompanies synapse maturation (3). Experimental denewation in aduHmiceleedsontheotherhandtoanimxeaseinmusdePAectivities (4). These musde PAS might then ba responsble for the degradstion of scme components of the muscle basement membrane (5), thereby setting the stage for reinnervation after denervation (6). In order to detem-rine the possible involvement of PAS in the basement membrane degradation associated with denervation observedinWcbMer,specificPAac6vitiesinbicepsandgasImcnemius musdeexbactswereestimatedusingbdhzymographyandasynthetic &&rate (B2251, K&i) relatively spsciticforplasmin. These activities were compared to those of the corresponding muscles in unaffeded mice (wr/+) from the same litter. PA activity was increased in Wcbbler mousemusclesversuscontrolmusdesandmoreinbicepsmusdethan in gastromemius musde, which is less inpaired by the mutation. Muscle PA was mostly uPA (f&=48 kD) more Ihan tPA (Mt=75 kD). In addiion, kinetlccharacteristics ofthe PA assay obtained with Wobbler mouse muscles lackedtheccnventbnal lag-phase,thus evidencing the presence of already formed plasmin. The neurological mutant mouse Wobbler is an autosomal recessive disorder that approximates the human lower motor neuron diseases (1) such as the fatal Amyotrophic Lateral Bderosis (ALS). The present results support the hypothesis of an activation of specific serine proteases in the pathogenesis of ALB (7). In additton, study of Wobbler PA regulaticn in muscle may shed light on the role of these cascades, and their inhibitors, in neuromuscular fomdon. 1. Andrewset al., Am. J. Path. 76,63-76. 1974. 2. La Vail and Irons, Brain Res. 463,78-89, 1988. 3. Hantai et al., Pm. Nati. Acad. Sci. USA, 86,38X366, 1989 4. Festofietal., J. Cell Biol. 193, 14161421, 1986. 5. Hantai and Festoff,Exp. Neural. 95,44-55.1987. 6. Hantai etal., Rev. Neural. (Paris) 144,660-667,1988. 7. Festoff,Med. Hypotheses6,121-131,198O.