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IGF-I REGULATION OF IN VITRO VASCULAR CALCIFICATION
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 for 1 and 2 weeks with DAR, CA IV activity increased significantly (Ctl : 11.39 ± 2.60, WVK : 12.24 ± 1.47, +DAR(5) : 29.46 ± 7,86, +DAR(5-6) : 35.44 ± 11.75 EU/mg of protein). Conclusion:Vascular mineral loss induced by the blockade of ET receptors seems to require the activation of a membrane-bound CA. Our results suggest that vascular mineral loss may proceed similarly as bone resorption, which may open new therapeutic strategies for ISH.
P526 AMLODIPINE PREVENTS AND REGRESSES AORTIC MEDIAL CALCIFICATION IN A RAT MODEL OF ISOLATED SYSTOLIC HYPERTENSION. Rachida Essalihi, Maarten Zandvliet, Huy Hao Dao, Pierre Moreau. Faculty of Pharmacy, University of Montreal, Montreal, Quebec, Canada. Objectives: Aortic medial calcification contributes to the development of isolated systolic hypertension (ISH). We have previously shown that an endothelin receptor antagonist (ETRA) can prevent and regress medial arterial calcification (MAC) and decrease pulse pressure (PP) in a rat model of ISH. Our propose was to determine if a calcium channel blocker (amlodipine), which inhibit some effects of endothelin, can also prevent and regress MAC. Methods: PREVENTION : Control (Ctl4) male Wistar rats were compared to rats receiving warfarin 15 mg/kg/day and vitamin k1 15 mg/kg/day (WVK4) alone or in association with amlodipine,15 mg/kg/day, WVK+ AMLO4) for 4 weeks. REGRESSION: Controls (Ctl8) rats were compared with rats treated only with W/K (WK8) during 8 weeks or in combination with amlodipine during the last 4 weeks (WVK8+AMLO4). Study endpoints were diastolic, systolic, mean blood pressure (DBP, SBP, MBP), PP, pulse wave velocity (PWV) and aortic and carotid calcium content. Results: Inactivation of matrix Gla protein by WVK for 4 or 8 weeks increased calcium content by 10 and 25 fold (P < .05) in the aorta and carotid arteries, respectivly. The treatment also induced a significant elevation of PP by a selective augmentation of SBP over DBP. As in ISH, MBP was unchanged and PWV was significatly elevated. Amlodipine prevented aortic MAC and the increase of PP but only partially blunted carotid calcification and the elevation of PWV. Moreover, amlodipine regressed aortic MAC but was unable to decrease significantly the carotid calcification. Nevertheless, amlodipine decreased PP at 8 weeks by decreasing SBP and increasing DBP. Conclusion: The clinical efficacy of the calcium channel blocker to treat ISH in the elderly could be explained by their beneficial effect on vascular calcification. It remains to be determined if this effect is related to the inhibition, by amlodipine, of the vascular effects of endothelin. Pfizer Canada
P527 THE ROLE OF TYPE III SODIUM-DEPENDENT PHOSPHATE COTRANSPORTER PIT-1 IN SMOOTH MUSCLE CELL CALCIFICATION. Xianwu Li, Cecilia M. Giachelli. University of Washington, Seattle, WA.
S185
arteries in chronic renal failure patients. Our previous studies demonstrated that elevated phosphate induces calcification of smooth muscle cells (SMC) and enhances the expression of the osteoblastic differentiation markers osteocalcin and Cbfa-1 in vitro. Phosphate enters the cell primarily through the sodium-dependent phosphate cotransporter on the membrane. Three types of sodium-dependent phosphate cotransporters have been identified so far. The type III, especially pit-1, is predominantly expressed in human SMC. We also showed previously that inhibition of phosphate transport by phosphonoformic acid blocks phosphate-induced calcification, suggesting sodium-dependent phosphate cotransporters may mediate phosphate’s effect. In the present study, we used the RNA interference approach to specifically knockdown the expression of pit-1 and to determine its role in the regulation of SMC calcification. The retroviral system was used to stably express pit-1 small interfering double stranded RNAs (siRNA) in human SMC. Northern blot analysis showed that the siRNA efficiently and specifically suppressed pit-1 gene expression without affecting other membrane transporters, such as the sodium-hydrogen antiporter. Expression of pit-1 siRNA caused a time- and concentration-dependent inhibition of sodium-dependent phosphate uptake. Furthermore, phosphate-induced SMC calcification was inhibited by pit-1 siRNA. Our studies are the first to demonstrate that pit-1 plays a critical role in phosphate-induced SMC calcification. NIH
P528 IGF-I REGULATION OF IN VITRO VASCULAR CALCIFICATION. Kristen Radcliff, Yin Tintut, Moeen Abedin, Jignesh Patel, Linda Demer. UCLA, Los Angeles, CA. Vascular calcification is clinically significant and occurs by a mechanism recapitulating osteogenesis. Serum levels of insulin-like growth factor-I (IGF), a known regulator of bone formation, correlate with reduced cardiac events. To determine whether it regulates vascular calcification in vitro, we tested the effects of IGF on a subpopulation of aortic smooth muscle cells known as calcifying vascular cells (CVC), which spontaneously differentiate into an osteoblast phenotype. Results showed that IGF significantly inhibited osteoblastic differentiation of the vascular cells, as indicated by reduced activity of the osteoblast marker, alkaline phosphatase (ALP) (32 + 2%). IGF also inhibited ALP induction by peroxide (84 + 6%), TNF-alpha (30 + 5%), and bacterial lipopolysaccharide (LPS, 41 + 5%). Insulin, a hormone closely related to IGF, had a similar inhibitory effect but VEGF did not. IGF increased proliferation in CVC (225 + 7%) as evidenced by 3H-thymidine incorporation. The inhibitory effects of IGF were not attenuated by blocking proliferation with a MEK inhibitor, PD98059 nor by blocking intracellular signaling with a PI3K inhibitor (LY294002), a p38 MAPK inhibitor (SB203580), or a tyrosine kinase inhibitor (Genistein). Lithium chloride, a known inhibitor of glycogen synthase kinase 3-beta (GSK), inhibited differentiation of CVC in an additive fashion with IGF (55 + 5%). These results suggest that IGF inhibits both the spontaneous and induced osteoblastic differentiation of CVC, and that the inhibition is through modulation of a GSK-dependent pathway. NIH R01 HL69261
Vascular calcification occurs in the patients with atherosclerosis, diabetes and renal failure. It is clearly associated with increased risk of cardiovascular morbidity and mortality. Approximately half of all death in end-stage renal disease patients is due to cardiovascular diseases. Recent evidence suggested that vascular calcification is an actively regulated process. A growing number of factors have been identified that either promote or inhibit this process. Clinical and animal studies indicate that elevated serum phosphate concentration is associated with vascular calcification as well as cardiovascular disease mortality. Hyperphosphatemia cause a progressive calcification in coronary arteries, mitral and aortic valves, and peripheral
P529 OSTEOPONTIN MEDIATED INHIBITION AND REGRESSION OF ECTOPIC CALCIFICATION. Rupak M. Rajachar, Cecilia M. Giachelli. Bioengineering-University of Washington, Seattle, WA. Until recently vascular calcification was regarded as an unregulated process. However evidence of bone gene expression in blood vessels coupled
P526 AMLODIPINE PREVENTS AND REGRESSES AORTIC MEDIAL CALCIFICATION IN A RAT MODEL OF ISOLATED SYSTOLIC HYPERTENSION. Rachida Essalihi, Maarten Zandvliet, Huy Hao Dao, Pierre Moreau. Faculty of Pharmacy, University of Montreal, Montreal, Quebec, Canada. Objectives: Aortic medial calcification contributes to the development of isolated systolic hypertension (ISH). We have previously shown that an endothelin receptor antagonist (ETRA) can prevent and regress medial arterial calcification (MAC) and decrease pulse pressure (PP) in a rat model of ISH. Our propose was to determine if a calcium channel blocker (amlodipine), which inhibit some effects of endothelin, can also prevent and regress MAC. Methods: PREVENTION : Control (Ctl4) male Wistar rats were compared to rats receiving warfarin 15 mg/kg/day and vitamin k1 15 mg/kg/day (WVK4) alone or in association with amlodipine,15 mg/kg/day, WVK+ AMLO4) for 4 weeks. REGRESSION: Controls (Ctl8) rats were compared with rats treated only with W/K (WK8) during 8 weeks or in combination with amlodipine during the last 4 weeks (WVK8+AMLO4). Study endpoints were diastolic, systolic, mean blood pressure (DBP, SBP, MBP), PP, pulse wave velocity (PWV) and aortic and carotid calcium content. Results: Inactivation of matrix Gla protein by WVK for 4 or 8 weeks increased calcium content by 10 and 25 fold (P < .05) in the aorta and carotid arteries, respectivly. The treatment also induced a significant elevation of PP by a selective augmentation of SBP over DBP. As in ISH, MBP was unchanged and PWV was significatly elevated. Amlodipine prevented aortic MAC and the increase of PP but only partially blunted carotid calcification and the elevation of PWV. Moreover, amlodipine regressed aortic MAC but was unable to decrease significantly the carotid calcification. Nevertheless, amlodipine decreased PP at 8 weeks by decreasing SBP and increasing DBP. Conclusion: The clinical efficacy of the calcium channel blocker to treat ISH in the elderly could be explained by their beneficial effect on vascular calcification. It remains to be determined if this effect is related to the inhibition, by amlodipine, of the vascular effects of endothelin. Pfizer Canada
P527 THE ROLE OF TYPE III SODIUM-DEPENDENT PHOSPHATE COTRANSPORTER PIT-1 IN SMOOTH MUSCLE CELL CALCIFICATION. Xianwu Li, Cecilia M. Giachelli. University of Washington, Seattle, WA.
S185
arteries in chronic renal failure patients. Our previous studies demonstrated that elevated phosphate induces calcification of smooth muscle cells (SMC) and enhances the expression of the osteoblastic differentiation markers osteocalcin and Cbfa-1 in vitro. Phosphate enters the cell primarily through the sodium-dependent phosphate cotransporter on the membrane. Three types of sodium-dependent phosphate cotransporters have been identified so far. The type III, especially pit-1, is predominantly expressed in human SMC. We also showed previously that inhibition of phosphate transport by phosphonoformic acid blocks phosphate-induced calcification, suggesting sodium-dependent phosphate cotransporters may mediate phosphate’s effect. In the present study, we used the RNA interference approach to specifically knockdown the expression of pit-1 and to determine its role in the regulation of SMC calcification. The retroviral system was used to stably express pit-1 small interfering double stranded RNAs (siRNA) in human SMC. Northern blot analysis showed that the siRNA efficiently and specifically suppressed pit-1 gene expression without affecting other membrane transporters, such as the sodium-hydrogen antiporter. Expression of pit-1 siRNA caused a time- and concentration-dependent inhibition of sodium-dependent phosphate uptake. Furthermore, phosphate-induced SMC calcification was inhibited by pit-1 siRNA. Our studies are the first to demonstrate that pit-1 plays a critical role in phosphate-induced SMC calcification. NIH
P528 IGF-I REGULATION OF IN VITRO VASCULAR CALCIFICATION. Kristen Radcliff, Yin Tintut, Moeen Abedin, Jignesh Patel, Linda Demer. UCLA, Los Angeles, CA. Vascular calcification is clinically significant and occurs by a mechanism recapitulating osteogenesis. Serum levels of insulin-like growth factor-I (IGF), a known regulator of bone formation, correlate with reduced cardiac events. To determine whether it regulates vascular calcification in vitro, we tested the effects of IGF on a subpopulation of aortic smooth muscle cells known as calcifying vascular cells (CVC), which spontaneously differentiate into an osteoblast phenotype. Results showed that IGF significantly inhibited osteoblastic differentiation of the vascular cells, as indicated by reduced activity of the osteoblast marker, alkaline phosphatase (ALP) (32 + 2%). IGF also inhibited ALP induction by peroxide (84 + 6%), TNF-alpha (30 + 5%), and bacterial lipopolysaccharide (LPS, 41 + 5%). Insulin, a hormone closely related to IGF, had a similar inhibitory effect but VEGF did not. IGF increased proliferation in CVC (225 + 7%) as evidenced by 3H-thymidine incorporation. The inhibitory effects of IGF were not attenuated by blocking proliferation with a MEK inhibitor, PD98059 nor by blocking intracellular signaling with a PI3K inhibitor (LY294002), a p38 MAPK inhibitor (SB203580), or a tyrosine kinase inhibitor (Genistein). Lithium chloride, a known inhibitor of glycogen synthase kinase 3-beta (GSK), inhibited differentiation of CVC in an additive fashion with IGF (55 + 5%). These results suggest that IGF inhibits both the spontaneous and induced osteoblastic differentiation of CVC, and that the inhibition is through modulation of a GSK-dependent pathway. NIH R01 HL69261
Vascular calcification occurs in the patients with atherosclerosis, diabetes and renal failure. It is clearly associated with increased risk of cardiovascular morbidity and mortality. Approximately half of all death in end-stage renal disease patients is due to cardiovascular diseases. Recent evidence suggested that vascular calcification is an actively regulated process. A growing number of factors have been identified that either promote or inhibit this process. Clinical and animal studies indicate that elevated serum phosphate concentration is associated with vascular calcification as well as cardiovascular disease mortality. Hyperphosphatemia cause a progressive calcification in coronary arteries, mitral and aortic valves, and peripheral
P529 OSTEOPONTIN MEDIATED INHIBITION AND REGRESSION OF ECTOPIC CALCIFICATION. Rupak M. Rajachar, Cecilia M. Giachelli. Bioengineering-University of Washington, Seattle, WA. Until recently vascular calcification was regarded as an unregulated process. However evidence of bone gene expression in blood vessels coupled