- Email: [email protected]
SERUM DERIVED FACTORS INHIBIT CONTRACTILITY OF ADULT RAT VENTRICULAR CARDIOMYOCYTES
S142
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
We conclude that low pH is a potent stimulus to release CGRP from the mouse heart. This effect seems to be primarily mediated through activation of TRPV1 receptors that are known to be expressed by slowly conducting, mainly nociceptive primary afferent nerve fibers. With respect to the potentially important vasodilatory and cardioprotective functions of CGRP, the role of CGRP secretion from primary afferents in myocardial ischemia and other cardiac disorders deserves further investigation.
P392 DELIVERY OF ENDOTHELIAL PAS DOMAIN PROTEIN 1 GENE PROMOTES MATURE ANGIOGENESIS THROUGH THE TRANSACTIVATION OF BOTH VEGF AND ITS RECEPTOR, FLT-1. Norihiko Takeda, Koji Maemura, Yasushi Imai, Daiji Kawanami, Tomohiro Harada, Takefumi Nojiri, Ryozo Nagai. Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo. Hypoxia plays an extremely important role in the pathogenesis of many cardiovascular diseases. In hypoxic conditions, each organ or cell shows compensatory response at the molecular and cellular levels. Hypoxia inducible factor-1a (HIF-1a) is expressed ubiquitously and serves as a master regulatory gene in the process of hypoxic response. Especially in the cardiovascular system, each organ promotes angiogenesis in order to increase the delivery of oxygen and nutrients. Endothelial PAS domain protein 1 (EPAS1) is a member of basic-helix-loop-helix/PAS domain containing transcription factor family and has a high homology to HIF-1a. In contrast to HIF-1a, EPAS1 is mainly expressed in vascular endothelial cells. Under hypoxic conditions, both HIF-1a and EPAS1 are reported to transactivate VEGF promoter. However, VEGF alone is not sufficient to assemble functional vasculature. Thus, in addition to VEGF, some other molecules are expected to contribute to the production of mature vessels. To elucidate the molecular mechanisms of angiogenesis in terms of vessel maturation, it may be beneficial to clarify the target genes of EPAS1 besides VEGF. In this study, using microarray technique, we searched target genes of EPAS1 in vascular endothelial cells and identified 130 genes including VEGF, adrenomedullin, and prostacyclin synthase. One of them is Flt-1, a receptor for VEGF, which is also mainly expressed in vascular endothelial cells. Reporter analysis using Flt-1 promoter showed EPAS1 transactivates Flt-1 promoter through HIF-1 binding site upstream of Flt-1 promoter. Gel shift assay confirmed that the heterodimer of EPAS1 and ARNT binds to this HIF-1 binding site. The expression of Flt-1 is upregulated during hypoxia, on the other hand, overexpression of dominant-negative EPAS1 partially attenuated the induction of endogeneous Flt-1 mRNA expression induced by hypoxia. Moreover, chemotactic assay showed adenovirus mediated overexpression of EPAS1 into human umbilical vein endothelial cells enhanced migratory response against VEGF. Finally, using mouse wound healing models, we found that adenovirus mediated delivery of EPAS1 gene increased the expression of VEGF, Flk-1, Flt-1 and Tie2 mRNA at the wound site and significantly promoted the wound healing process. Also, EPAS1 increased capillary density at the wound site. Furthermore the newly formed vessels were surrounded by smooth muscle a_actin positive mural cells. In conclusion, EPAS1 promotes Flt-1 gene expression and induces mRNA expression of VEGF, Flk-1, and Tie2 leading to enhancement of angiogenesis in vivo. Thus EPAS1 may contribute to the construction of mature vessels by modulating the coordinated expressions of VEGF, Flt-1, Flk-1, and Tie2.
P393 EXPRESSION PATTERNS OF VEGF AND VEGF RECEPTORS IN ISCHAEMIC SKELETAL MUSCLE OF PATIENTS AFTER LIMB AMPUTATION. Vincent van Weel, Kees van Leuven, Esther Kuiper, Reinier Schlingemann, Hajo van Bockel, Victor van Hinsbergh, Paul Quax. Gaubius Laboratory TNO-PG, Leiden, the Netherlands, Dept. Ophtalmology, Amsterdam Medical Center, Amsterdam, the Netherlands, Dept. Ophtalmology, Amsterdam Medical Center, Amsterdam, the Netherlands, Dept. Surgery, Leiden University Medical Center, Leiden, the Netherlands, Dept. Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam the Netherlands. Vascular Endothelial Growth Factor (VEGF) (gene) therapy has been shown to promote angiogenesis in various animal models. Recent placebo-controlled trials using VEGF (gene) therapy in patients with arterial occlusive disease did not show an improved clinical outcome. We hypothesized that administration of VEGF is not effective, because VEGF is already abundantly expressed in ischaemic tissues of these patients. Endogenous protein expression of VEGF and its receptors was studied in amputated limbs of 15 patients with peripheral artery disease. For this, skeletal muscle biopsies were performed at 3 levels of the amputated limb, namely at the amputation level (representing non-ischaemic tissue), distal soleus muscle (increased ischaemia), and, most distally, interossius muscle between the toes (most severe ischaemia). Immunohistochemistry of skeletal muscle sections were performed for VEGF-A, B, C and D, VEGF receptor 1, 2 and 3, CD31 and CD34. Protein expression at different levels was compared in a single-blinded fashion. Statistical differences in expression were determined using the Sign-test. Anti-CD31- and anti-CD34stained vessel density en size was determined by computer analysis of 10 images per section. Both VEGF-A and B were significantly elevated in ischaemic muscle as compared to non-ischaemic muscle ( p = .049). Both proteins were especially located in the cytoplasm of muscle fibers. However, there was no expression of VEGF-C detectable. VEGF-D was abundantly expressed in muscle fibers throughout the limb, however not selectively elevated in ischaemic muscle ( p = .23). Of the receptors, only VEGF receptor 2 was significantly elevated in ischaemic muscle ( p = .003). VEGF receptor 1 and 2 were expressed both on endothelial cells and surrounding muscle fibers, VEGF receptor 3 expression was restricted to the endothelium. Endothelial staining showed a significant increase of vessel density and size in ischaemic versus non-ischaemic muscle ( p = < .01). In conclusion, VEGF A, B and D, not C, are abundantly expressed in skeletal muscle of patients with chronic peripheral artery disease. In addition, VEGF A and B expression are elevated in ischaemic versus non-ischaemic muscle, suggesting ischaemia-induced upregulation. Correspondingly, vessel density and size was significantly increased in the ischaemic region, suggesting an angiogenic response. However, this angiogenic response was not sufficient for limb salvage. It is unlikely that administration of additional VEGF (alone) can improve clinical outcome in these patients. Adding growth factors that more selectively enhance collateral artery growth may stimulate blood flow more efficiently.
P394 SERUM DERIVED FACTORS INHIBIT CONTRACTILITY OF ADULT RAT VENTRICULAR CARDIOMYOCYTES. Hubert Walinski, Yingjin Wang, Bruce McManus, Thomas Podor. James Hogg Research Laboratories/iCAPTUR4E Centre for Cardiovascular and Pulmonary Research, Department of Pathology and Laboratory Medicine, St. Paul’s Hospital-University of British Columbia, Vancouver, BC, Canada. Background: We have recently discovered a possible mechanism by which vitronectin (VN) may modulate post-myocardial infarction (MI) ventricular contractility. Studies illustrate specific binding of vitronectin to vimentin and desmin intermediate filaments (IFs) in the sarcomeres of
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 perturbed cardiomyocytes ischemia/reperfused (I/R) hearts. Here we have examined in cultured adult rat cardiomyocytes whether transient hypoxia exposes the IF contractile apparatus to serum-derived factors such as (VN) and leads to impaired contractility. Hypothesis: Plasma VN serves a cardioprotective effect by binding IFs on damaged cardiomyocytes, thus limiting energy demands by interfering with IF-dependent control of cardiomyocyte contractility. Methods: Cardiomyocytes were isolated from adult rat hearts using a Langendorff perfusion apparatus and medium containing collagenase and bovine serum albumin (BSA). Cardiomyocytes were plated in the presence or absence of 10% fetal bovine serum in M199 media containing 1% BSA and either with 10% fetal bovine serum (FBS) or without, then incubated at 37 °C overnight. Half of the cells cultured in either serum or serum free media were subjected to transient hypoxia using nitrogen gas (95%) for 30 minutes and the other half remained normoxic. Cardiomyocytes ( n = 80) were then electrically stimulated (40 mV) and contractile distance measured using the soft edge detection system (IonWizard) following which fractional shortening and shortening velocity were determined. Cardiomyocytes from each group were fixed with peraformaldehyde, permeabilized using Triton-X 100 and subjected to immunofluorescence and confocal microscopy. Results: There was a significant decrease in fractional shortening in hypoxic versus normoxic cardiomyocytes in both the serum free (29.00%, p = .00025) and the FBS group (27.97%, p = .0012). A substantial decline was also observed in the shortening velocities, (21.18%, p = .0418) and (17.32%, p = .1043) respectively. There was also a notable decrease in fractional shortening of hypoxic cardiomyocytes in the presence of FBS (16.95 ± 1.33 versus 13.70 ± 1.19, p = .065). An apparent reduction in shortening velocity was also perceived between the two hypoxic groups (41.43 ± 4.04 and 34.85 ± 3.07, p = .187). Confocal analysis illustrated the positive staining for VN in the cells subjected to hypoxia and serum, but was absent from both groups under serum free conditions and in the FBS normoxic group. Also, recent preliminary data indicates that purified serum-derived VN significantly inhibits fractional shortening and shortening velocity in hypoxia treated cardiomyocytes in a dose-dependent manner. Conclusion: Serum factors such as VN enter damaged cardiomyocytes and inhibit contractility, potentially by binding to sarcomeric IF cytoskeleton of the hypoxic cells. VN may thereby exert a cardioprotective role by limiting energy use. CIHR
P395 EXPRESSION OF HYPOXIA-INDUCED MITOGENIC FACTOR (HIMF) IN CULTURED MOUSE VASCULAR ENDOTHELIAL CELLS AND MOUSE ISCHEMIC TISSUE. Yifu Zhou, Dechun Li, Susan L. Balenger, Xingwu Teng, Roger A Johns. Vascular Biology Laboratory, Dept. ACCM, Johns Hopkins University, Baltimore, MD 21205. Background: Found in inflammatory zone1 (FIZZ1) or resistin-like molecule a´ (RELMa´) is a cysteine-rich secreted protein, associated with pulmonary inflammation. We recently found that hypoxia upregulated FIZZ1/RELMa´ expression in pulmonary vasculature, and FIZZ1/RELMa´ showed potent mitogenic, angiogenic and vasoconstrictive effects. Therefore, it was renamed as hypoxia-induced mitogenic factor (HIMF). In the current studies, we investigated the endogenous hypoxia-induced expression of HIMF in cultured mouse vascular endothelial cells and ischemic mouse tissue. Methods and Results: Mouse vascular endothelial cells (SVEC-4), cultured in T-75 flasks, were subjected to either normal culture conditions (37 °C, 5% CO2 , 20% O2) or hypoxia (1% O2) for 4, 6, 8, and 10 hrs, and 1, 2, 3, 4, and 5 days. The expression of HIMF was then tested by Western blotting using specific anti-mouse HIMF antibody. Under normal culture conditions, a low level of HIMF protein was detected in SVEC-4 cells;
S143
hypoxia increased the expression to twice that in normal conditions starting at 4 hrs, and maintained the increased level for three additional days of hypoxia. On days 4 and 5, the protein levels had returned to baseline when cells were still subject to hypoxic conditions. No detectable levels of HIMF protein were found in conditioned medium. Left femoral arteries of C57/ BL6 mice were ligated to create unilateral hind limb ischemia. At various time points (6 hrs, 1, 3, 5, 7, 14, and 28 days) following ligation, tissues from ischemic areas were collected and lysed for Western blot analysis. The expression of HIMF was then tested by using specific anti-mouse HIMF antibody. Recombinant HIMF protein was used as positive control. We found that after sham surgery and 6hrs after ligation, HIMF expression was slightly increased compared with normal tissues. However, from 1 to 7 days, HIMF expression dramatically increased, suggesting that it may be involved in the angiogenesis process. Conclusions: HIMF is constitutively expressed in mouse vascular endothelial cells and hindlimb tissues. Its expression dramatically increased under hypoxic and ischemic conditions. The in vitro and in vivo tests will be valuable for studying the regulation of HIMF expression and the role of HIMF in physiologic and pathophysiologic processes such as angiogenesis, vasculogenesis and collateral vessel formation. The study was funded by NIH grant RO-1 HL 39706
Oxidative Stress and Vascular Function P396 INVOLVEMENT OF HYDROGEN PEROXIDE IN THE ENDOTHELIUM-DEPENDENT CONTRACTION OF RAT RENAL ARTERY TO ACETYLCHOLINE. Yu-Jing Gao, Robert M.K.W. Lee. Department of Anesthesia McMaster University, Hamilton, Ontario, Canada. In addition to the release of relaxing factors such as endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone through the release of endothelium-derived contracting factor(s) (EDCF). The identity of the EDCF in the renal artery is not known. In this study, the involvement of reactive oxygen species in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh) was investigated. ACh (10-5 M) induced a transient contraction of rat renal artery with intact endothelium, but not in the artery with endothelium removed. Nitric oxide synthase inhibitor (e.g., NG-nitro – Larginine) enhanced the contraction to ACh. Treatment with catalase greatly reduced the EDC to ACh, while the contraction to phenylephrine or to KCl was not affected. Cyclooxygenase inhibitor (diclofenac), thromboxane A2/prostaglandine H2 receptor antagonist (SQ 29548) nearly abolished EDC. ACh enhanced the production of hydrogen peroxide (detected by Amplex Red) in the renal artery with intact endothelium. We suggest that hydrogen peroxide is probably a modulator of EDC in renal artery in response to Ach, through its stimulation in the production of thromboxane A2 by endothelium. Heart and Stroke Foundation of Ontario, Canada
P397 VASCULAR EFFECTS OF A GENE VARIANT OF EXTRACELLULAR SUPEROXIDE DISMUTASE IN SPONTANEOUSLY HYPERTENSIVE RATS. Abdullah Alwahdani, Yi Chu, Robert M. Brooks, Donald D. Lund, Frank M. Faraci, Donald D. Heistad. University of Iowa, Iowa City, Iowa, University of Iowa and VAMC, Iowa City, Iowa. We reported recently that gene transfer of extracellular superoxide dismutase (ECSOD) reduces arterial pressure, and improves vascular and renal
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
We conclude that low pH is a potent stimulus to release CGRP from the mouse heart. This effect seems to be primarily mediated through activation of TRPV1 receptors that are known to be expressed by slowly conducting, mainly nociceptive primary afferent nerve fibers. With respect to the potentially important vasodilatory and cardioprotective functions of CGRP, the role of CGRP secretion from primary afferents in myocardial ischemia and other cardiac disorders deserves further investigation.
P392 DELIVERY OF ENDOTHELIAL PAS DOMAIN PROTEIN 1 GENE PROMOTES MATURE ANGIOGENESIS THROUGH THE TRANSACTIVATION OF BOTH VEGF AND ITS RECEPTOR, FLT-1. Norihiko Takeda, Koji Maemura, Yasushi Imai, Daiji Kawanami, Tomohiro Harada, Takefumi Nojiri, Ryozo Nagai. Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo. Hypoxia plays an extremely important role in the pathogenesis of many cardiovascular diseases. In hypoxic conditions, each organ or cell shows compensatory response at the molecular and cellular levels. Hypoxia inducible factor-1a (HIF-1a) is expressed ubiquitously and serves as a master regulatory gene in the process of hypoxic response. Especially in the cardiovascular system, each organ promotes angiogenesis in order to increase the delivery of oxygen and nutrients. Endothelial PAS domain protein 1 (EPAS1) is a member of basic-helix-loop-helix/PAS domain containing transcription factor family and has a high homology to HIF-1a. In contrast to HIF-1a, EPAS1 is mainly expressed in vascular endothelial cells. Under hypoxic conditions, both HIF-1a and EPAS1 are reported to transactivate VEGF promoter. However, VEGF alone is not sufficient to assemble functional vasculature. Thus, in addition to VEGF, some other molecules are expected to contribute to the production of mature vessels. To elucidate the molecular mechanisms of angiogenesis in terms of vessel maturation, it may be beneficial to clarify the target genes of EPAS1 besides VEGF. In this study, using microarray technique, we searched target genes of EPAS1 in vascular endothelial cells and identified 130 genes including VEGF, adrenomedullin, and prostacyclin synthase. One of them is Flt-1, a receptor for VEGF, which is also mainly expressed in vascular endothelial cells. Reporter analysis using Flt-1 promoter showed EPAS1 transactivates Flt-1 promoter through HIF-1 binding site upstream of Flt-1 promoter. Gel shift assay confirmed that the heterodimer of EPAS1 and ARNT binds to this HIF-1 binding site. The expression of Flt-1 is upregulated during hypoxia, on the other hand, overexpression of dominant-negative EPAS1 partially attenuated the induction of endogeneous Flt-1 mRNA expression induced by hypoxia. Moreover, chemotactic assay showed adenovirus mediated overexpression of EPAS1 into human umbilical vein endothelial cells enhanced migratory response against VEGF. Finally, using mouse wound healing models, we found that adenovirus mediated delivery of EPAS1 gene increased the expression of VEGF, Flk-1, Flt-1 and Tie2 mRNA at the wound site and significantly promoted the wound healing process. Also, EPAS1 increased capillary density at the wound site. Furthermore the newly formed vessels were surrounded by smooth muscle a_actin positive mural cells. In conclusion, EPAS1 promotes Flt-1 gene expression and induces mRNA expression of VEGF, Flk-1, and Tie2 leading to enhancement of angiogenesis in vivo. Thus EPAS1 may contribute to the construction of mature vessels by modulating the coordinated expressions of VEGF, Flt-1, Flk-1, and Tie2.
P393 EXPRESSION PATTERNS OF VEGF AND VEGF RECEPTORS IN ISCHAEMIC SKELETAL MUSCLE OF PATIENTS AFTER LIMB AMPUTATION. Vincent van Weel, Kees van Leuven, Esther Kuiper, Reinier Schlingemann, Hajo van Bockel, Victor van Hinsbergh, Paul Quax. Gaubius Laboratory TNO-PG, Leiden, the Netherlands, Dept. Ophtalmology, Amsterdam Medical Center, Amsterdam, the Netherlands, Dept. Ophtalmology, Amsterdam Medical Center, Amsterdam, the Netherlands, Dept. Surgery, Leiden University Medical Center, Leiden, the Netherlands, Dept. Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam the Netherlands. Vascular Endothelial Growth Factor (VEGF) (gene) therapy has been shown to promote angiogenesis in various animal models. Recent placebo-controlled trials using VEGF (gene) therapy in patients with arterial occlusive disease did not show an improved clinical outcome. We hypothesized that administration of VEGF is not effective, because VEGF is already abundantly expressed in ischaemic tissues of these patients. Endogenous protein expression of VEGF and its receptors was studied in amputated limbs of 15 patients with peripheral artery disease. For this, skeletal muscle biopsies were performed at 3 levels of the amputated limb, namely at the amputation level (representing non-ischaemic tissue), distal soleus muscle (increased ischaemia), and, most distally, interossius muscle between the toes (most severe ischaemia). Immunohistochemistry of skeletal muscle sections were performed for VEGF-A, B, C and D, VEGF receptor 1, 2 and 3, CD31 and CD34. Protein expression at different levels was compared in a single-blinded fashion. Statistical differences in expression were determined using the Sign-test. Anti-CD31- and anti-CD34stained vessel density en size was determined by computer analysis of 10 images per section. Both VEGF-A and B were significantly elevated in ischaemic muscle as compared to non-ischaemic muscle ( p = .049). Both proteins were especially located in the cytoplasm of muscle fibers. However, there was no expression of VEGF-C detectable. VEGF-D was abundantly expressed in muscle fibers throughout the limb, however not selectively elevated in ischaemic muscle ( p = .23). Of the receptors, only VEGF receptor 2 was significantly elevated in ischaemic muscle ( p = .003). VEGF receptor 1 and 2 were expressed both on endothelial cells and surrounding muscle fibers, VEGF receptor 3 expression was restricted to the endothelium. Endothelial staining showed a significant increase of vessel density and size in ischaemic versus non-ischaemic muscle ( p = < .01). In conclusion, VEGF A, B and D, not C, are abundantly expressed in skeletal muscle of patients with chronic peripheral artery disease. In addition, VEGF A and B expression are elevated in ischaemic versus non-ischaemic muscle, suggesting ischaemia-induced upregulation. Correspondingly, vessel density and size was significantly increased in the ischaemic region, suggesting an angiogenic response. However, this angiogenic response was not sufficient for limb salvage. It is unlikely that administration of additional VEGF (alone) can improve clinical outcome in these patients. Adding growth factors that more selectively enhance collateral artery growth may stimulate blood flow more efficiently.
P394 SERUM DERIVED FACTORS INHIBIT CONTRACTILITY OF ADULT RAT VENTRICULAR CARDIOMYOCYTES. Hubert Walinski, Yingjin Wang, Bruce McManus, Thomas Podor. James Hogg Research Laboratories/iCAPTUR4E Centre for Cardiovascular and Pulmonary Research, Department of Pathology and Laboratory Medicine, St. Paul’s Hospital-University of British Columbia, Vancouver, BC, Canada. Background: We have recently discovered a possible mechanism by which vitronectin (VN) may modulate post-myocardial infarction (MI) ventricular contractility. Studies illustrate specific binding of vitronectin to vimentin and desmin intermediate filaments (IFs) in the sarcomeres of
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 perturbed cardiomyocytes ischemia/reperfused (I/R) hearts. Here we have examined in cultured adult rat cardiomyocytes whether transient hypoxia exposes the IF contractile apparatus to serum-derived factors such as (VN) and leads to impaired contractility. Hypothesis: Plasma VN serves a cardioprotective effect by binding IFs on damaged cardiomyocytes, thus limiting energy demands by interfering with IF-dependent control of cardiomyocyte contractility. Methods: Cardiomyocytes were isolated from adult rat hearts using a Langendorff perfusion apparatus and medium containing collagenase and bovine serum albumin (BSA). Cardiomyocytes were plated in the presence or absence of 10% fetal bovine serum in M199 media containing 1% BSA and either with 10% fetal bovine serum (FBS) or without, then incubated at 37 °C overnight. Half of the cells cultured in either serum or serum free media were subjected to transient hypoxia using nitrogen gas (95%) for 30 minutes and the other half remained normoxic. Cardiomyocytes ( n = 80) were then electrically stimulated (40 mV) and contractile distance measured using the soft edge detection system (IonWizard) following which fractional shortening and shortening velocity were determined. Cardiomyocytes from each group were fixed with peraformaldehyde, permeabilized using Triton-X 100 and subjected to immunofluorescence and confocal microscopy. Results: There was a significant decrease in fractional shortening in hypoxic versus normoxic cardiomyocytes in both the serum free (29.00%, p = .00025) and the FBS group (27.97%, p = .0012). A substantial decline was also observed in the shortening velocities, (21.18%, p = .0418) and (17.32%, p = .1043) respectively. There was also a notable decrease in fractional shortening of hypoxic cardiomyocytes in the presence of FBS (16.95 ± 1.33 versus 13.70 ± 1.19, p = .065). An apparent reduction in shortening velocity was also perceived between the two hypoxic groups (41.43 ± 4.04 and 34.85 ± 3.07, p = .187). Confocal analysis illustrated the positive staining for VN in the cells subjected to hypoxia and serum, but was absent from both groups under serum free conditions and in the FBS normoxic group. Also, recent preliminary data indicates that purified serum-derived VN significantly inhibits fractional shortening and shortening velocity in hypoxia treated cardiomyocytes in a dose-dependent manner. Conclusion: Serum factors such as VN enter damaged cardiomyocytes and inhibit contractility, potentially by binding to sarcomeric IF cytoskeleton of the hypoxic cells. VN may thereby exert a cardioprotective role by limiting energy use. CIHR
P395 EXPRESSION OF HYPOXIA-INDUCED MITOGENIC FACTOR (HIMF) IN CULTURED MOUSE VASCULAR ENDOTHELIAL CELLS AND MOUSE ISCHEMIC TISSUE. Yifu Zhou, Dechun Li, Susan L. Balenger, Xingwu Teng, Roger A Johns. Vascular Biology Laboratory, Dept. ACCM, Johns Hopkins University, Baltimore, MD 21205. Background: Found in inflammatory zone1 (FIZZ1) or resistin-like molecule a´ (RELMa´) is a cysteine-rich secreted protein, associated with pulmonary inflammation. We recently found that hypoxia upregulated FIZZ1/RELMa´ expression in pulmonary vasculature, and FIZZ1/RELMa´ showed potent mitogenic, angiogenic and vasoconstrictive effects. Therefore, it was renamed as hypoxia-induced mitogenic factor (HIMF). In the current studies, we investigated the endogenous hypoxia-induced expression of HIMF in cultured mouse vascular endothelial cells and ischemic mouse tissue. Methods and Results: Mouse vascular endothelial cells (SVEC-4), cultured in T-75 flasks, were subjected to either normal culture conditions (37 °C, 5% CO2 , 20% O2) or hypoxia (1% O2) for 4, 6, 8, and 10 hrs, and 1, 2, 3, 4, and 5 days. The expression of HIMF was then tested by Western blotting using specific anti-mouse HIMF antibody. Under normal culture conditions, a low level of HIMF protein was detected in SVEC-4 cells;
S143
hypoxia increased the expression to twice that in normal conditions starting at 4 hrs, and maintained the increased level for three additional days of hypoxia. On days 4 and 5, the protein levels had returned to baseline when cells were still subject to hypoxic conditions. No detectable levels of HIMF protein were found in conditioned medium. Left femoral arteries of C57/ BL6 mice were ligated to create unilateral hind limb ischemia. At various time points (6 hrs, 1, 3, 5, 7, 14, and 28 days) following ligation, tissues from ischemic areas were collected and lysed for Western blot analysis. The expression of HIMF was then tested by using specific anti-mouse HIMF antibody. Recombinant HIMF protein was used as positive control. We found that after sham surgery and 6hrs after ligation, HIMF expression was slightly increased compared with normal tissues. However, from 1 to 7 days, HIMF expression dramatically increased, suggesting that it may be involved in the angiogenesis process. Conclusions: HIMF is constitutively expressed in mouse vascular endothelial cells and hindlimb tissues. Its expression dramatically increased under hypoxic and ischemic conditions. The in vitro and in vivo tests will be valuable for studying the regulation of HIMF expression and the role of HIMF in physiologic and pathophysiologic processes such as angiogenesis, vasculogenesis and collateral vessel formation. The study was funded by NIH grant RO-1 HL 39706
Oxidative Stress and Vascular Function P396 INVOLVEMENT OF HYDROGEN PEROXIDE IN THE ENDOTHELIUM-DEPENDENT CONTRACTION OF RAT RENAL ARTERY TO ACETYLCHOLINE. Yu-Jing Gao, Robert M.K.W. Lee. Department of Anesthesia McMaster University, Hamilton, Ontario, Canada. In addition to the release of relaxing factors such as endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone through the release of endothelium-derived contracting factor(s) (EDCF). The identity of the EDCF in the renal artery is not known. In this study, the involvement of reactive oxygen species in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh) was investigated. ACh (10-5 M) induced a transient contraction of rat renal artery with intact endothelium, but not in the artery with endothelium removed. Nitric oxide synthase inhibitor (e.g., NG-nitro – Larginine) enhanced the contraction to ACh. Treatment with catalase greatly reduced the EDC to ACh, while the contraction to phenylephrine or to KCl was not affected. Cyclooxygenase inhibitor (diclofenac), thromboxane A2/prostaglandine H2 receptor antagonist (SQ 29548) nearly abolished EDC. ACh enhanced the production of hydrogen peroxide (detected by Amplex Red) in the renal artery with intact endothelium. We suggest that hydrogen peroxide is probably a modulator of EDC in renal artery in response to Ach, through its stimulation in the production of thromboxane A2 by endothelium. Heart and Stroke Foundation of Ontario, Canada
P397 VASCULAR EFFECTS OF A GENE VARIANT OF EXTRACELLULAR SUPEROXIDE DISMUTASE IN SPONTANEOUSLY HYPERTENSIVE RATS. Abdullah Alwahdani, Yi Chu, Robert M. Brooks, Donald D. Lund, Frank M. Faraci, Donald D. Heistad. University of Iowa, Iowa City, Iowa, University of Iowa and VAMC, Iowa City, Iowa. We reported recently that gene transfer of extracellular superoxide dismutase (ECSOD) reduces arterial pressure, and improves vascular and renal