Biomedical subjects

H Haller

Publications and source records attributed to H Haller.

At least 109 records · Page 6Linked to original sources

PubMed1999

Differential expression of protein kinase C isoforms in streptozotocin-induced diabetic rats.

BACKGROUND: The cellular effects of hyperglycemia are mediated by protein kinase C (PKC). However, PKC consists of several distinct isoforms, and their contribution to the pathogenesis of diabetic complications in different organs is not clear. We investigated the expression and translocation of PKC isoforms alpha, betaI, betaII, delta, epsilon, and zeta in kidney, heart, and aorta from diabetic rats. METHODS: Hyperglycemia was induced with streptozotocin (70 mg/kg) in the rat. After four weeks, PKC isoform expression was assessed by Western blot after tissue fractionation and by immunohistochemistry. RESULTS: Streptozotocin increased blood glucose from 117.0 +/- 3.6 to 510.0 +/- 19.4 mg/dl (N = 8, P < 0.01) and induced albuminuria. PKC isoforms alpha, beta, delta, epsilon, and zeta were all detected in control animals. Western blot showed increased PKC alpha expression in kidney and heart (160% and 170%, respectively). PKC betaI, betaII, and delta expression was not influenced by hyperglycemia. PKC zeta was decreased in diabetic animals in both tissues by 60%. The membrane association of PKC alpha and PKC epsilon was increased; however, the relative amount of PKC in the particulate fraction was not influenced by hyperglycemia. Immunohistochemistry revealed a marked increase in PKC alpha immunoreactivity in renal glomeruli and interstitial capillaries, cardiac capillaries, and skeletal muscle, as well as in the endothelial cells of larger arteries. PKC beta showed a small decrease in the glomeruli. PKC epsilon was increased in renal tubules in diabetic rats but was decreased in the myocardium. PKC zeta was expressed in both myocardial and glomerular cells but was decreased during hyperglycemia. Our results demonstrate that PKC isoforms are differentially regulated in kidney and heart in diabetes. High glucose increases PKC alpha expression, whereas PKC zeta is down-regulated. The finding that PKC alpha is mostly increased in endothelial cells supports a role for PKC alpha in functional endothelial disturbances observed in diabetes.

Animals↗

PubMed1999

Regulation of MAC-1 (CD11b/CD18) expression on circulating granulocytes in endurance runners.

PURPOSE: We tested the hypothesis that degranulation of granulocytes and upregulation of the granulocyte integrin MA-1 (CD11b/CD18) are related to exercise duration and/or intensity. We also investigated whether or not the expression of MAC-1 would be influenced by body temperature or dehydration. Moreover, we tested the hypothesis that changes in leukocyte counts and changes in MAC1 expression with endurance exercise are independently regulated. METHODS: In eight amateur runners, MAC-1 (CD11b/CD18) surface expression on granulocytes was determined by fluorescent antibody cell sorting, before and after an incremental maximal treadmill test, a moderate 3-h run, and a competitive marathon race. RESULTS: Expression CD11b on granulocytes was increased by 10+/-9.6% (P < 0.05) after the maximal treadmill test and by 84+/-76% (P < 0.01) after the marathon run. There was no change in CD11b expression after the moderate 3-h run. CD18 expression was not significantly changed after any of the exercise protocols. CONCLUSION: Expression of CD11b on granulocytes is increased with intense endurance exercise, either incremental maximal treadmill testing or competitive marathon running, but not in moderate endurance training. Thus, exhaustive exercise may be one mechanism for the upregulation of integrin adhesive receptors on granulocytes. This phenomenon could be in part responsible for increased adhesion of granulocytes to endothelial cells and could facilitate tissue infiltration after endurance exercise.

Adult↗

PubMed1999

Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import.

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha7. To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin alpha proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha3, whereas most of the other substrates were imported by all importin alpha proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin alpha proteins were revealed when two substrates were offered simultaneously.

Acetyltransferases↗

PubMed1999

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta.

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.

Calcium↗

PubMed1999

Urokinase activates the Jak/Stat signal transduction pathway in human vascular endothelial cells.

Endothelial cells demonstrate high urokinase expression and upregulation of urokinase receptors in response to vascular injury. Urokinase receptor binding facilitates endothelial cell migration into an arterial wound; however, the signaling cascade induced by the urokinase receptor in this cell type is incompletely understood. Because the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway seems to be important for vessel function, we investigated the hypothesis that urokinase receptor binding activates Jak/Stat signaling in human vascular endothelial cells. Incubation of endothelial cells with urokinase-type plasminogen activator (uPA,1 nmol/L) induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with a molecular weight between 80 to 90 and 130 to 140 kDa. The same pattern of tyrosine phosphorylation was found after treatment with 1 nmol/L ATF, the urokinase amino-terminal fragment, which is devoid of proteolytic activity but still binds to the urokinase receptor. Using coimmunoprecipitation techniques, we demonstrated that the activated urokinase receptor is associated with 2 cytoplasmic tyrosine kinases of the Jak family, viz, Jak1 and Tyk2. uPA and ATF induced a time-dependent activation of both kinases, as shown by immunoprecipitation and Western blot analysis. Using electrophoretic mobility shift and supershift assays, we then demonstrated that Stat1 is rapidly activated in endothelial cells in response to uPA and ATF. Furthermore, Stat1 specifically binds to the regulatory elements interferon-gamma activation site/interferon-stimulated response element. The uPA-induced, time-dependent translocation of Stat1 to cell nuclei was confirmed by confocal microscopy study and immunoblotting of nuclear extracts with an anti-Stat1 antibody. This study provides evidence for a novel signaling pathway for uPA in human vascular endothelial cells. Direct activation of the Jak/Stat system via the uPA-receptor complex may be an important mechanism for endothelial cell migration and/or proliferation during angiogenesis and after vascular injury.

Biological Transport↗

PubMed1999

Farnesol blocks the L-type Ca2+ channel by targeting the alpha 1C subunit.

We recently demonstrated that farnesol, a 15-carbon isoprenoid, blocks L-type Ca2+ channels in vascular smooth muscle cells. To elucidate farnesol's mechanism of action, we performed whole-cell and perforated-patch clamp experiments in rat aortic A7r5 cells and in Chinese hamster ovary (CHO) C9 cells expressing smooth muscle Ca2+ channel alpha 1C subunits. Farnesol dose-dependently and voltage-independently inhibited Ba2+ currents in both A7r5 and CHOC9 cells, with similar half-maximal inhibitions at 2.6 and 4.3 micromol/L, [corrected] respectively (P=NS). In both cell lines, current inhibition by farnesol was prominent over the whole voltage range without changes in the current-voltage relationship peaks. Neither intracellular infusion of the stable GDP analogue guanosine-5'-O-(2-thiodiphosphate) (100 micromol/L) [corrected] via the patch pipette nor strong conditioning membrane depolarization prevented the inhibitory effect of farnesol, which indicates G protein-independent inhibition of Ca2+ channels. In an analysis of the steady-state inactivation curve for voltage dependence, farnesol induced a significant, negative shift ( approximately 10 mV) of the potential causing 50% channel inactivation in both cell lines (P<0. 001). In contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unlike pharmacological Ca2+ channel blockers, farnesol blocked Ca2+ currents in the resting state: initial block was 63+/-8% in A7r5 cells and 50+/-9% in CHOC9 cells at a holding potential of -80 mV. We then gave 500 mg/kg body weight farnesol by gavage to Sabra hypertensive and normotensive rats and found that farnesol reduced blood pressure significantly in the hypertensive strain for at least 48 hours. We conclude that farnesol may represent an endogenous smooth muscle L-type Ca2+ channel antagonist. Because farnesol is active in cells expressing only the pore-forming alpha1 subunit, the data further suggest that this subunit represents the molecular target for farnesol binding and principal action. Finally, farnesol has a blood pressure-lowering action that may be relevant in vivo.

Animals↗

PubMed1999

Endothelial cell dysfunction and arterial wall hypertrophy are associated with disturbed carbohydrate metabolism in patients at risk for cardiovascular disease.

To investigate the effects of fasting and postprandial glucose on endothelial cell function and intima-media thickness, we studied 60 men with cardiovascular risk factors. Postischemic, endothelium-dependent vasodilatation was measured after 3 minutes of ischemia at the radial artery with high-resolution echo tracking. Common carotid artery intima-media thickness was measured by B-mode ultrasound. Glucose tolerance was determined by a 75-g oral glucose load. Fasting glucose levels were inversely correlated with postischemic, endothelium-dependent vasodilatation (r=-0.24, P<0.05) and directly correlated with intima-media thickness (r=0.26, P<0.05). However, postischemic, endothelium-dependent vasodilatation and intima-media thickness were not correlated. All subjects with normal postischemic, endothelium-dependent vasodilatation also had a normal intima-media thickness, whereas some subjects with impaired postischemic, endothelium-dependent vasodilatation also had a normal intima-media thickness. Multiple regression analysis revealed a profound influence of age on intima-media thickness to the exclusion of all other variables. The same age-adjusted analysis for postischemic, endothelium-dependent vasodilatation accepted fasting glucose, followed by 2-hour postprandial glucose, as variables, but no others. Subjects with fasting glucose values >100 mg/dL showed reduced postischemic, endothelium-dependent vasodilatation (59 versus 120 microm, P<0.05) and a higher intima-media thickness (right: 0.76 versus 0.62 mm, P<0.05; left: 0.78 versus 0.63 mm, P<0. 05) compared with those with fasting glucose values <100 mg/dL. Subjects with 2-hour postprandial glucose values >125 mg/dL had no reduced postischemic, endothelium-dependent vasodilatation compared with subjects with a 2-hour postprandial glucose <125 mg/dL; however, their intima-media thickness (right: 0.66 versus 0.62 mm; left: 0. 68 versus 0.62 mm; P<0.05 for both) was greater. Thus, high fasting rather than postprandial glucose values are associated with both postischemic, endothelium-dependent vasodilatation and increased intima-media thickness. Postischemic endothelium-dependent vasodilatation may precede increased intima-media thickness.

Adult↗

PubMed1999

Hypertension-induced end-organ damage : A new transgenic approach to an old problem.

Angiotensin (Ang) II-induced organ damage has fascinated students of hypertension since the work of Wilson and Byrom. We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the vascular wall accompanies PAI-1, MCP-1, and VEGF expression. The expression of TGF-beta and deposition of extracellular matrix proteins follows, which is accompanied by fibrinoid vasculitis in small vessels of the heart and kidneys. Angiotensin-converting enzyme inhibitors and AT1 receptor blockers each lowered blood pressure and shifted pressure natriuresis partially leftward by different mechanisms. When combined, they normalized blood pressure, pressure natriuresis, and protected from vasculopathy completely. Renin inhibition lowered blood pressure partially, but protected from vasculopathy completely. Endothelin receptor blockade had no influence on blood pressure but protected from vasculopathy and improved survival. We show evidence that Ang II stimulates oxidative stress directly or indirectly via endothelin 1 and that NFkappaB is upregulated in this model. We speculate that the transcription factors NFkappaB and AP-1 are involved with initiating chemokine and cytokine expression, leading to the above cascade. The unique model and our pharmacological probes will enable us to test these hypotheses.

Angiotensin II↗

PubMed1999

Monocyte infiltration and adhesion molecules in a rat model of high human renin hypertension.

Hypertension and kidney damage in the double transgenic rat (dTGR) harboring both human renin and human angiotensinogen genes are dependent on the human components of the renin angiotensin system. We tested the hypothesis that monocyte infiltration and increased adhesion molecule expression are involved in the pathogenesis of kidney damage in dTGR. We also evaluated the effects of long-term angiotensin-converting enzyme (ACE) inhibition, AT1 blockade, and human renin inhibition on monocyte recruitment and inflammatory response in dTGR. Systolic blood pressure and 24-hour albuminuria were markedly increased in 7-week-old dTGR as compared with age-matched normotensive Sprague Dawley rats. We found a significant monocyte/macrophage infiltration in the renal perivascular space and increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the interstitium, intima, and adventitia of the small renal vessels. alphaLbeta2 integrin and alpha4beta1 integrin, the corresponding ligands for ICAM-1 and VCAM-1, were also found on infiltrating monocytes/macrophages. The expression of plasminogen activator inhibitor-1 and fibronectin in the kidneys of dTGR were increased and distributed similarly to ICAM-1. In 4-week-old dTGR, long-term treatment with ACE inhibition (cilazapril), AT1 receptor blockade (valsartan), and human renin inhibition (RO 65-7219) (each drug 10 mg/kg by gavage once a day for 3 weeks) completely prevented the development of albuminuria. However, only cilazapril and valsartan were able to decrease blood pressure to normotensive levels. Interestingly, the drugs were all equally effective in preventing monocyte/macrophage infiltration and the overexpression of adhesion molecules, plasminogen activator inhibitor-1, and fibronectin in the kidney. Our findings indicate that angiotensin II causes monocyte recruitment and vascular inflammatory response in the kidney by blood pressure-dependent and blood pressure-independent mechanisms. ACE inhibition, AT1 receptor blockade, and human renin inhibition all prevent monocyte/macrophage infiltration and increased adhesion molecule expression in the kidneys of dTGR.

Angiotensinogen↗

PubMed1999

Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor.

Immune mechanisms and the renin-angiotensin system are implicated in preeclampsia. We investigated 25 preeclamptic patients and compared them with 12 normotensive pregnant women and 10 pregnant patients with essential hypertension. Antibodies were detected by the chronotropic responses to AT1 receptor-mediated stimulation of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists. Immunoglobulin from all preeclamptic patients stimulated the AT1 receptor, whereas immunoglobulin from controls had no effect. The increased autoimmune activity decreased after delivery. Affinity-column purification and anti-human IgG and IgM antibody exposure implicated an IgG antibody directed at the AT1 receptor. Peptides corresponding to sites on the AT1 receptor's second extracellular loop abolished the stimulatory effect. Western blotting with purified patient IgG and a commercially obtained AT1 receptor antibody produced bands of identical molecular weight. Furthermore, confocal microscopy of vascular smooth muscle cells showed colocalization of purified patient IgG and AT1 receptor antibody. The protein kinase C (PKC) inhibitor calphostin C prevented the stimulatory effect. Our results suggest that preeclamptic patients develop stimulatory autoantibodies against the second extracellular AT1 receptor loop. The effect appears to be PKC-mediated. These novel autoantibodies may participate in the angiotensin II-induced vascular lesions in these patients.

Amino Acid Sequence↗

PubMed1999

Intracellular actions of angiotensin II in vascular smooth muscle cells.

Angiotensin II (AngII) is present inside vascular smooth muscle cells (VSMC); however, its intracellular functions, if any, are unknown. AngII was administered by microinjection. AngII was identified in endosomes and in the nucleus. Microinjection of AngII (10(-10) M) led to a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i) in the cytosol and in the nucleus. The [Ca2+]i increase was the result of an influx of extracellular Ca2+ ions. The intracellular AngII effect was totally inhibited by concomitant injection of the AngII type 1 receptor blocker candesartan. Desensitization of extracellular AngII receptors, on the other hand, did not influence the intracellular effects, and neither did extracellular candesartan. The increase in [Ca2+]i was observed not only in the microinjected cell but also in directly adjacent VSMC. In contrast to the microinjected cells, the [Ca2+]i increase in the adjacent cells was mostly the result of Ca2+ release from intracellular stores. Pretreatment with thapsigargin, which interferes with Ca2+ release from intracellular stores, abolished the AngII response in adjacent cells. Microinjection of inositol trisphosphate induced a [Ca2+]i response in adjacent cells that was similar to the AngII-induced effects. Preincubation of VSMC with uncoupling substances did not decrease the AngII response but prevented a [Ca2+]i surge in adjacent cells. Tyrosine phosphorylation was next examined. Phosphorylation was detected in the injected cells, primarily in the cytoskeleton. It can be concluded that intracellular AngII binds to intracellular AngII receptors and elicits increased [Ca2+]i in the injected cell and then in cells in the immediate neighborhood. Cell-cell contact is necessary for the AngII-mediated effects. These data suggest that intracellular AngII may stimulate a cluster of VSMC from a single cell, via the release of second messengers.

Angiotensin II↗

PubMed1998

Endothelial-cell permeability and protein kinase C in pre-eclampsia.

BACKGROUND: Oedema and vascular leakage play a part in the pathogenesis of pre-eclampsia. We tested the hypothesis that serum from pre-eclamptic patients increases endothelial-cell permeability and examined possible signal-transduction pathways. METHODS: We studied eight patients with pre-eclampsia, eight normotensive pregnant women, eight non-pregnant women, five pregnant patients with pre-existing hypertension, and four hypertensive non-pregnant women. Cultured human umbilical-vein endothelial-cell monolayers were used and permeability was measured by albumin flux. The part played by protein kinase C (PKC) signalling was examined by down-regulation with phorbol ester and with the inhibitors Goe 6976 and staurosporine. PKC isoforms were assessed by western blot and confocal microscopy. Antisense oligodesoxynucleotides (ODN) were used to test for specific PKC isoforms. FINDINGS: Serum from pre-eclamptic women increased endothelial permeability significantly (by 100%, p<0.01). The change in permeability decreased rapidly after delivery. Serum from normotensive pregnant women and non-pregnant women had no effect. Permeability was not influenced by serum from patients with essential hypertension or pregnant patients with pre-existing hypertension. Serum from pre-eclamptic patients induced a translocation of PKC isoforms alpha and epsilon within the cells. Goe 6976 and staurosporine (10(-8) mol/L) inhibited the increase in permeability induced by serum from pre-eclamptic patients. Down-regulation of PKC alpha and, to a lesser extent, PKC epsilon by antisense ODN also inhibited the pre-eclampsia-induced permeability increase. INTERPRETATION: Serum from pre-eclamptic patients contains a factor or factors that increase endothelial-cell permeability. The effect of pre-eclamptic serum may be mediated by PKC alpha and epsilon.

Adult↗

PubMed1998

Integrin-induced protein kinase Calpha and Cepsilon translocation to focal adhesions mediates vascular smooth muscle cell spreading.

The extracellular matrix influences the cellular spreading of vascular smooth muscle cells (VSMCs) via integrin receptors. However, the intracellular signaling mechanisms are still incompletely understood. We investigated the hypothesis that VSMCs binding to fibronectin activates the protein kinase C (PKC) pathway, causes differential intracellular PKC isoform translocation, and mediates cell spreading. VSMCs binding to poly-L-lysine or preincubated with Arg-Gly-Asp (RGD) peptides were used as controls. Diacylglycerol (DAG) and phospholipase D (PLD) activity were measured by thin-layer chromatography. Intracellular distribution of PKC isoforms was assessed by confocal microscopy. VSMCs binding to fibronectin induced focal adhesions and cell spreading within 30 minutes. Fibronectin induced a rapid increase in DAG content, peaking at 10 minutes with a sustained response for <1 hour. In contrast, PLD activity was not influenced by specific binding to fibronectin. PKC isoforms alpha, delta, epsilon, and zeta were assessed by confocal microscopy. Fibronectin induced a PKC isoform translocation to the cell nucleus and to focal adhesions within minutes. The nuclear PKCalpha immunoreactivity was transiently increased. PKC isoforms a and epsilon were both translocated to focal adhesions. The intracellular distributions of other PKC isoforms were not influenced by fibronectin. The effects of fibronectin on DAG generation, the translocation of PKCalpha and PKCepsilon, and cell spreading were all abolished by the incubation with RGD peptides. Downregulation of PKC isoforms alpha and epsilon with specific antisense oligodinucleotides resulted in a significant inhibition of cell spreading. Our results show that integrins induce intracellular signaling in VSMCs via DAG and PKC. PKC isoform a is translocated to the nucleus, whereas PKC isoforms alpha and epsilon are translocated to focal adhesions. Both isoforms seem to play a role in inside-out integrin signaling and cell spreading.

Animals↗

PubMed1998

The Jak/Stat pathway and urokinase receptor signaling in human aortic vascular smooth muscle cells.

The binding of urokinase plasminogen activator (uPA) to its specific receptor (uPAR) facilitates migration of vascular smooth muscle cells (VSMC). However, the signaling cascade utilized by the urokinase receptor is only incompletely understood. We investigated intracellular uPA/uPAR signaling in human aortic VSMC from the cell membrane to the nucleus. uPA binding to VSMC induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with molecular masses of 53-60, 85-90, and 130-140 kDa. By using co-immunoprecipitation techniques and in vitro kinase assays, the uPAR-associated proteins were identified as Janus (Jak) and Src non-receptor protein-tyrosine kinases (PTK) Jak1, Tyk2, and p59(fyn), p53/56(lyn), p53/59(hck), and p55(fgr). Furthermore, uPA induced a time-dependent reversible translocation of the Stat1 (signal transducer and activator of transcription) protein to the VSMC nuclei, as shown by confocal microscopy studies. Using an electrophoretic mobility shift assay, we then demonstrated that Stat1 is rapidly activated in response to stimulation with uPA and specifically binds to the DNA regulatory elements GAS (interferon-gamma activation site) and ISRE (interferon-stimulated response element). Mobility supershift experiments confirmed DNA-protein complexes containing Stat1 protein. Migration experiments with double immunofluorescence staining revealed polarization of uPAR, and colocalization with Jak1 and Tyk2 to the leading edge of the migrating cells. Under the same conditions, Jak2, Jak3, and the Src-PTKs remained randomly distributed over the entire body of the cells. Our studies therefore suggest that, in VSMC, the uPAR-signaling complex utilizes at least two different mechanisms, a direct signaling pathway utilizing the Jak/Stat cascade and a second signal transduction mechanism via Src-like protein-tyrosine kinases. uPA-induced signaling via Jak/Stat is most likely involved in the regulation of cell migration, while the functional purpose of the uPA-associated Src-PTK activation remains to be elucidated.

Cell Movement↗

PubMed1998

Angiotensin II acts intracellularly in vascular smooth muscle cells.

Angiotensin (ANG) II is present inside vascular smooth muscle cells (VSMC); however, its intracellular functions if any, are unknown. We tested the hypothesis that intracellular Ang II exerts effects on cytosolic calcium [Ca++]i in VSMC. ANG II was administered via microinjection. Microinjection of ANG II led to a rapid increase in [Ca++]i in the cytosol and in the nucleus. The [Ca++]i increase was due to the influx of extracellular Ca++ ions. The intracellular ANG II effect was totally inhibited by the concomitant injection of the ANG II antagonist CV-11947. Desensitization of extracellular ANG II receptors on the other hand, did not influence the intracellular effects, nor did extracellular CV-11947. The increase in [Ca++]i was not only observed in the microinjected cell, but also in directly adjacent VSMC. In contrast to the microinjected cells, the [Ca++]i increase in the adjacent cells was mostly due to release from intracellular stores. Pretreatment with thapsigargin abolished the ANG II response in adjacent cells. Microinjection of IP3 induced a [Ca++]i response in adjacent cells which was similar to the ANG II-induced effects. Preincubation of VSMC with the uncoupling substances DMSO and heptanol did not decrease the ANG II response, but instead prevented a [Ca++]i surge in adjacent cells. We conclude that intracellular ANG II binds to intracellular ANG II receptors and elicits an increased [Ca++]i in the injected cell and thereafter cells in the immediate neighborhood. Cell-cell contact is necessary for the ANG II-mediated effects. The data suggest that ANG II-related effects may be amplified by an intracellular action.

Angiotensin II↗

PubMed1998

The clinical importance of postprandial glucose.

Atherosclerotic lesions develop over a long period of time and result from complex changes in the arterial wall. Although these changes are not fully understood, there is much evidence to suggest that elevated plasma glucose levels contribute to the development of atherosclerotic lesions. Many studies have shown that there is a strong correlation between elevated plasma glucose levels and the risk of developing cardiovascular disease. Effects of glucose on the arterial wall include immediate effects, which occur rapidly in response to elevated plasma glucose levels, and long-term effects, which result from non-enzymatic glycosylation of various proteins. These adverse effects of elevated plasma glucose levels suggest that tight control of blood glucose levels in patients with diabetes could possibly reduce the risk of cardiovascular complications. This is borne out by the results of clinical studies in patients with type 1 diabetes. Therapy to reduce blood glucose levels may also be appropriate in individuals with impaired glucose tolerance, as this condition is associated with postprandial hyperglycaemia and a significant risk of developing cardiovascular disease.

Arteriosclerosis↗

PubMed1998

Intracellular targeting and protein kinase C in vascular smooth muscle cells: specific effects of different membrane-bound receptors.

Protein kinase C is an important second messenger system, which is translocated from the cytosol to the cell membrane upon cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with different agonists. First, we analysed the effects of angiotensin II and platelet-derived growth factor (PDGF). Confocal microscopy showed a rapid assembly of PKC alpha along cytosolic fibres followed by a translocation towards the nucleus with angiotensin II. PDGF engendered a similar, but much slower response; however, a cytoskeletal distribution was not observed. We then investigated the effects of thrombin and bFGF on nuclear translocation. bFGF induced a rapid translocation of the isoform towards the perinuclear region and into the nucleus. bFGF had a similar effect on PKC epsilon. In contrast, thrombin had a smaller effect on nuclear translocation of PKC alpha and did not influence PKC epsilon, but instead induced a rapid nuclear translocation of PKC zeta. Thus, tyrosine kinase receptor activation via bFGF induces a rapid association of PKC alpha and epsilon within nuclear structures. Our results show that agonists cause, not only a translocation of protein kinase C isoforms into the cell membrane but also into the cell nucleus. Lastly, we analyzed the nuclear immunoreactivity of the PKC isoforms, alpha, delta, epsilon and zeta in vascular smooth muscle cells during the cell cycle. Resting cells were stimulated with foetal calf serum (FCS, 10%), which translocated PKC alpha and epsilon to the perinuclear region and into the nucleus, while PKC delta and zeta showed no increase in nuclear immunoreactivity. After 4 h of FCS, the nuclear immunoreactivity for PKC alpha and epsilon was reduced to or below control values. At 8 h, increased nuclear expression of isoforms alpha, epsilon and zeta was observed, while isoform delta was not affected. Our results demonstrate a complex spatial and temporal regulation of PKC isoforms in response to vasoactive hormones and growth factors. We suggest that protein kinase C may be important for nuclear signaling and demonstrate that nuclear translocation of PKC isoforms is differentially regulated during the cell cycle.

Angiotensin II↗

PubMed1998

ICAM-1 antisense oligodesoxynucleotides prevent reperfusion injury and enhance immediate graft function in renal transplantation.

BACKGROUND: Ischemia-reperfusion injury after organ transplantation is a major cause of delayed graft function. We showed earlier that antisense oligodesoxynucleotides (ODN) for intercellular adhesion molecule-1 (ICAM-1) ameliorate reperfusion injury after acute ischemia. This study tested the hypothesis that antisense ODN for ICAM-1 prevents ischemia-reperfusion injury and facilitates immediate graft function in a rat autotransplantation model. METHODS: Both kidneys were removed from male Lewis rats and re-implanted the left kidney after 30 minutes of cold ischemia time. The warm ischemia time was 60 minutes. Sham operated, uninephrectomized animals served as controls for renal function and histology. ICAM-1 antisense ODN (5 mg/kg), reverse ODN, or saline-vehicle were administered to donor animals i.v. six hours before autotransplantation. Glomerular filtration rate (insulin clearance), and serum creatinine concentrations were measured 24 hours post-transplantation. Tubular necrosis severity was assessed by histological grading scale. ICAM-1 expression was determined by immunohistochemistry and Western blot. RESULTS: Antisense ODN decreased ICAM-1 expression and leukocyte infiltration significant. Antisense ODN-treated animals showed significantly less tubular necrosis, than controls. Serum creatinine of antisense ODN-treated animals (N = 6) was 0.55 +/- 0.02 mg/dl compared to 1.92 +/- 0.07 mg/dl in reverse ODN-treated controls (N = 6; P < 0.01), 24 hours after transplantation. Antisense ODN-treated animals had normal GFR (0.93 +/- 0.07 ml/min/kidney wt) compared to sham-operated animals (0.95 +/- 0.09 ml/min/kidney wt), while autotransplanted animals treated with reverse ODN or saline-vehicle were all anuric. The ischemia-reperfusion-induced up-regulation of MHC class II was totally prevented by antisense ODN. CONCLUSIONS: ICAM-1 inhibition ameliorates ischemia-reperfusion injury and prevents delayed graft function. Antisense ODN-treatment of donors or donor organs for ICAM-1 may be useful for the prevention of reperfusion injury in human renal transplantation.

Animals↗