1,25-Dihydroxyvitamin D3 stimulates vascular smooth muscle cell proliferation through a VEGF-mediated pathway
2006; Elsevier BV; Volume: 69; Issue: 8 Linguagem: Inglês
10.1038/sj.ki.5000304
ISSN1523-1755
AutoresAna Cardus Figueras, Eva Parisi, Carme Gallego, Martí Aldea, Elvira Fernández, José Manuel Valdivielso,
Tópico(s)Thyroid and Parathyroid Surgery
ResumoAtherosclerosis is a complex process characterized by an increase in the wall thickness owing to the accumulation of cells and extracellular matrix between the endothelium and the smooth muscle cell wall. This process is associated with different pathologies and it is accelerated in patients with chronic renal failure. In these patients, decreased synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) leads to secondary complications, like hyperparathyroidism, and treatment with 1,25(OH)2D3 is a common practice. The effect of 1,25(OH)2D3 on vascular smooth muscle cells (VSMCs) calcification has been widely studied, but the role of 1,25(OH)2D3 on VSMC proliferation remains obscure. We have analyzed the effects of 1,25(OH)2D3 in the proliferation of VSMC. We found that 1,25(OH)2D3 (5–100 nM) induces a dose-dependent increase in VSMC proliferation in quiescent cells and in cells stimulated to grow. This increase in proliferation is achieved by shortening the G1 phase. The effect of 1,25(OH)2D3 on VSMC proliferation is mediated by an increase of the expression of vascular endothelial growth factor A (VEGF), as the inhibition of VEGF activity totally blunted the 1,25(OH)2 D3-induced VSMC proliferation. We found this increase in proliferation in vitro, ex vivo in aortic rings incubated with 1,25(OH)2D3, and in vivo in animals with a model of chronic renal failure (5/6 nephrectomy) treated with 1,25(OH)2D3 (1 μg/kg three times a week for 8 weeks). Thus, we conclude that 1,25(OH)2D3 induces increases in VSMC proliferation through an increase on VEGF expression. Atherosclerosis is a complex process characterized by an increase in the wall thickness owing to the accumulation of cells and extracellular matrix between the endothelium and the smooth muscle cell wall. This process is associated with different pathologies and it is accelerated in patients with chronic renal failure. In these patients, decreased synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) leads to secondary complications, like hyperparathyroidism, and treatment with 1,25(OH)2D3 is a common practice. The effect of 1,25(OH)2D3 on vascular smooth muscle cells (VSMCs) calcification has been widely studied, but the role of 1,25(OH)2D3 on VSMC proliferation remains obscure. We have analyzed the effects of 1,25(OH)2D3 in the proliferation of VSMC. We found that 1,25(OH)2D3 (5–100 nM) induces a dose-dependent increase in VSMC proliferation in quiescent cells and in cells stimulated to grow. This increase in proliferation is achieved by shortening the G1 phase. The effect of 1,25(OH)2D3 on VSMC proliferation is mediated by an increase of the expression of vascular endothelial growth factor A (VEGF), as the inhibition of VEGF activity totally blunted the 1,25(OH)2 D3-induced VSMC proliferation. We found this increase in proliferation in vitro, ex vivo in aortic rings incubated with 1,25(OH)2D3, and in vivo in animals with a model of chronic renal failure (5/6 nephrectomy) treated with 1,25(OH)2D3 (1 μg/kg three times a week for 8 weeks). Thus, we conclude that 1,25(OH)2D3 induces increases in VSMC proliferation through an increase on VEGF expression. Patients with chronic kidney disease (CKD) and dialysis patients show an increased mortality despite measures to optimize the dialysis treatment.1.Raine A.E.G. Margreiter R. Brunner F.P. et al.Report on management of renal-failure in Europe, Xxii, 1991.Nephrol Dial Transplant. 1992; 7: 7-35PubMed Google Scholar, 2.Levin A. Cardiac disease in chronic kidney disease: current understandings and opportunities for change.Blood Purif. 2004; 22: 21-27Crossref PubMed Scopus (12) Google Scholar One of the causes of this increase in mortality seems to be related to changes in the cardiovascular system.3.Amann K. Rychlik I. Miltenberger-Milteny G. et al.Left ventricular hypertrophy in renal failure.Kidney Int. 1998; 54: S78-S85Abstract Full Text Full Text PDF Scopus (88) Google Scholar, 4.Foley R.N. Parfrey P.S. Anemia in predialysis chronic renal failure: what are we treating?.J Am Soc Nephrol. 1998; 9: S82-S84PubMed Google Scholar Lindner et al.5.Lindner A. Charra B. Sherrard D.J. et al.Accelerated atherosclerosis in prolonged maintenance hemodialysis.N Engl J Med. 1974; 290: 697-701Crossref PubMed Scopus (1464) Google Scholar concluded that accelerated atherosclerosis, with proliferation of vascular smooth muscle cells (VSMCs), was the major cause of this increased cardiovascular mortality. However, in patients with CKD, another arteriopathy has been described, characterized by an increase in the thickness of the arterial wall with intimal proliferation and endovascular fibrosis with calcification.6.Amann K. Wolf B. Nichols C. et al.Aortic changes in experimental renal failure – hyperplasia or hypertrophy of smooth muscle cells?.Hypertension. 1997; 29: 770-775Crossref PubMed Scopus (92) Google Scholar, 7.Essary L.R. Wick M.R. Cutaneous calciphylaxis – an underrecognized clinicopathologic entity.Am J Clin Pathol. 2000; 113: 280-287Crossref PubMed Google Scholar This arteriosclerotic process leads to a stiffening of the arteries, abnormal coronary perfusion, and left ventricular hypertrophy.8.London G.M. Left ventricular hypertrophy: why does it happen?.Nephrol Dial Transplant. 2003; 18: 2-6Crossref Google Scholar Thus, unregulated proliferation of VSMC may play a central role in both arteriopathies. Under normal circumstances, VSMCs have very reduced proliferation rates, but different physiopathological stimuli can induce their growth.9.Berk B.C. Vascular smooth muscle growth: autocrine growth mechanisms.Physiol Rev. 2001; 81: 999-1030Crossref PubMed Scopus (317) Google Scholar A number of factors have been involved in the calcification process,10.Speer M.Y. Giachelli C.M. Regulation of cardiovascular calcification.Cardiovasc Pathol. 2004; 13: 63-70Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 11.Parhami F. Bostrom K. Watson K. et al.Role of molecular regulation in vascular calcification.J Atheroscler Thromb. 1996; 3: 90-94Crossref PubMed Scopus (44) Google Scholar but the mediators involved in VSMC proliferation are unknown. In CKD, impaired production of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a major contributor to the occurrence of parathyroid hyperplasia and to the increased synthesis and secretion of parathyroid hormone. Thus, 1,25(OH)2D3 is widely used to treat secondary hyperparathyroidism. Several studies have demonstrated that an excess of 1,25(OH)2D3 is arteriotoxic12.Rajasree S. Rajpal K. Kartha C.C. et al.Serum 25-hydroxyvitamin D-3 levels are elevated in South Indian patients with ischemic heart disease.Eur J Epidemiol. 2001; 17: 567-571Crossref PubMed Scopus (64) Google Scholar and that 1,25(OH)2D3 induces vascular calcifications in humans and experimental animals.13.Bajwa G.S. Morrison L.M. Ershoff B.H. Induction of aortic and coronary athero-arteriosclerosis in rats Fed A Hypervitaminosis-D, cholesterol-containing diet.Proc Soc Exp Biol Med. 1971; 138: 975Crossref PubMed Scopus (49) Google Scholar, 14.Liu L.B. Taylor C.B. Peng S.K. et al.Experimental arteriosclerosis in rhesus-monkeys induced by multiple risk-factors – cholesterol, vitamin-D, and nicotine.Paroi Arterielle-Arterial Wall. 1979; 5: 25-31PubMed Google Scholar Thus, the effects of 1,25(OH)2D3 on vascular calcification have been widely studied, but its effects on vascular cell proliferation remain unclear. Furthermore, the presence of vitamin D receptor (VDR) in endothelial cells and VSMCs, and the finding that endothelial cells synthesize 1,25(OH)2D3 raises new questions about its paracrine role.15.Merke J. Milde P. Lewicka S. et al.Identification and regulation of 1,25-dihydroxyvitamin-D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin-D3 – studies in cultured bovine aortic endothelial-cells and human dermal capillaries.J Clin Invest. 1989; 83: 1903-1915Crossref PubMed Scopus (280) Google Scholar, 16.Zehnder D. Bland R. Chana R.S. et al.Synthesis of 1,25-dihydroxyvitamin D-3 by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion.J Am Soc Nephrol. 2002; 13: 621-629Crossref PubMed Google Scholar Recent studies suggest that 1,25(OH)2D3 may have further implications in the atherosclerotic process. In this line of reasoning, Zhender et al.16.Zehnder D. Bland R. Chana R.S. et al.Synthesis of 1,25-dihydroxyvitamin D-3 by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion.J Am Soc Nephrol. 2002; 13: 621-629Crossref PubMed Google Scholar postulated that the synthesis of 1,25(OH)2D3 by endothelial cells has a paracrine/autocrine function and acts at a local level promoting leukocyte adhesion. Furthermore, Rebsamen et al.17.Rebsamen M.C. Sun J.X. Norman A.W. et al.1 Alpha, 25-dihydroxyvitamin D-3 induces vascular smooth muscle cell migration via activation of phosphatidylinositol 3-kinase.Circ Res. 2002; 91: 17-24Crossref PubMed Scopus (138) Google Scholar found that 1,25(OH)2D3 induced a dose-dependent increase in VSMC migration. Although it is known that 1,25(OH)2D3 acts on many cell lines to decrease the proliferation rate, the few studies aimed at analyzing the effect of 1,25(OH)2D3 on VSMCs showed controversial results.18.Maccarthy E.P. Yamashita W. Hsu A. et al.1,25-Dihydroxyvitamin-D3 and rat vascular smooth-muscle cell-growth.Hypertension. 1989; 13: 954-959Crossref PubMed Google Scholar, 19.Koh E. Morimoto S. Fukuo K. et al.1,25-Dihydroxyvitamin-D3 binds specifically to rat vascular smooth-muscle cells and stimulates their proliferation in vitro.Life Sci. 1988; 42: 215-223Crossref PubMed Scopus (54) Google Scholar Therefore, we decided to investigate whether 1,25(OH)2D3 exerts an effect on VSMC proliferation in vitro, in aortic rings ex vivo, and in a model of CKD in rats. We also analyzed the mediators involved in this process. We investigated the effect of crescent concentrations of 1,25(OH)2D3 (5–100 nM) on proliferation of serum-starved VSMCs. 1,25(OH)2D3 stimulated VSMC proliferation in a dose-dependent manner (Figure 1a). To determine if 1,25(OH)2D3 could increase the proliferative effect induced by growth factors, quiescent cells were treated for 24 h with basic fibroblast growth factor (bFGF) (20 ng/ml) with increasing concentrations of 1,25(OH)2D3 (5–100 nM). Figure 1b shows that 1,25(OH)2D3 increased proliferation in a dose-dependent manner, with significant stimulatory effects at doses as low as 5 nM. Then, we tested the effect of fetal bovine serum (FBS) addition to the 1,25(OH)2D3-induced proliferation. As expected, FBS had a strong effect on the proliferation and, at high concentrations, FBS was able to mask the effects mediated by 1,25(OH)2D3 (Figure 1c). We decided to perform flow cytometry analysis to further characterize the effect of 1,25(OH)2D3 on VSMC proliferation by bFGF. Fluorescence-activated cell sorter analysis showed an increase in the percentage of cells in S phase in the 1,25(OH)2D3-treated culture, indicating that 1,25(OH)2D3 advanced S-phase entry by 6 h (Figure 2). Then, we examined the effect of 1,25(OH)2D3 on VSMC proliferation in aortic rings ex vivo (Figure 3). 1,25(OH)2D3 induced an increase in proliferating cells with respect to the control. These cells were identified as VSMCs by labeling for α-actin. Finally, the effect of 1,25(OH)2D3in vivo was assessed in a model of CKD. Animals with 5/6 renal mass reduction and treated with 1,25(OH)2D3 for 8 weeks did not gain weight during the treatment (200±24 g), whereas animals treated with saline increased their weight (280±20 g). Total calcium levels were significantly increased in animals receiving 1,25(OH)2D3 compared with control animals (control: 11.02±0.14 mg/dl; 1,25(OH)2D3: 12.14±0.3 mg/dl. P<0.01). We also observed an increase in phosphate levels that did not reach statistical significance (control: 6.06±0.27 mg/dl; 1,25(OH)2D3: 6.68±0.35 mg/dl). In addition, levels of calcium phosphorous product (CaXPO4) product were significantly higher in rats receiving 1,25(OH)2D3 compared with control animals (control: 66.83±3 mg2/dl2; 1,25(OH)2D3: 81.3±5.2 mg2/dl2; P<0.01). Animals with 5/6 renal mass reduction presented a decreased creatinine clearance (control: 1.6±0.19; 5/6 nephrectomy: 1.047±0.17 ml/min). Treatment with 1,25(OH)2D3 further aggravated the renal failure (5/6 nephrectomy+1,25(OH)2 D3: 0.58±0.09 ml/min; P<0.05 vs 5/6 nephrectomy). Animals with 5/6 renal mass reduction and treated with 1,25(OH)2D3 for 8 weeks had a higher number of proliferative nuclei (stained for Ki67) than animals untreated (3J). The proliferative cells were identified as VSMC by α-actin immunofluorescence. Treatment with 1,25(OH)2D3 significantly increased the VDR mRNA levels in VSMC (Figure 4a). Results of the Western blot analysis showed that VDR protein increased by more than 10-fold after 12 h of treatment (Figure 4b). In Figure 4, we show the effects of 1,25(OH)2D3 treatment in VSMC on vascular endothelial growth factor A (VEGF) mRNA and protein expression. We found that treatment with 1,25(OH)2D3 significantly increased the levels of VEGF mRNA and protein (Figure 4c and d). The expression of fms-like tyrosine kinase 1 (FLT-1) receptor was slightly increased by real-time polymerase chain reaction (PCR) and Western blot (Figure 4e and f). No expression of FLK-1 was detected either by real-time PCR or Western Blot. To determine if an increase in VEGF was responsible for the increase in proliferation, we incubated VSMC with 1,25(OH)2D3 and a VEGF receptor antagonist (VGA1102). In Figure 5a, we can see that co-incubation with VGA1102 totally blunted the proliferative effect induced by 1,25(OH)2 D3. No toxic effect was detected in VSMC at the dose used (Figure 5b). We also incubated VSMC with 1,25(OH)2D3 and VEGF-neutralizing antibody. The addition of the antibody eliminated the increase in proliferation induced by 1,25(OH)2D3 (Figure 5c). The biological actions of 1,25(OH)2D3 are mediated by its specific receptor, the VDR.20.Minghetti P.P. Norman A.W. 1,25(Oh)2-vitamin-D3 receptors – gene-regulation and genetic circuitry.FASEB J. 1988; 2: 3043-3053Crossref PubMed Scopus (429) Google Scholar Therefore, to study the effect of 1,25(OH)2D3 in VSMCs, first we determined whether VSMCs show a functional and coherent response to vitamin D. It has been described that VDR levels are upregulated by 1,25(OH)2D3.21.Beckman M.J. Horst R.L. Reinhardt T.A. et al.Up-regulation of the intestinal 1,25-dihydroxyvitamin-D receptor during hypervitaminosis-D – a comparison between vitamin-D2 and vitamin-D3.Biochem Biophys Res Commun. 1990; 169: 910-915Crossref PubMed Scopus (18) Google Scholar We have found that 1,25(OH)2D3 increased VDR mRNA and protein levels in VSMC, indicating that the VDR pathway is fully functional in these cells. The content of VDR is a very important factor in the transcriptional activity in target cells. An increase in VDR number could 'sensitize' vascular cells to vitamin D action, as it occurs in other tissues (parathyroid glands, bowel mucosa, etc.).22.Cross H.S. Bareis P. Hofer H. et al.25-Hydroxyvitamin D(3)-1alpha-hydroxylase and vitamin D receptor gene expression in human colonic mucosa is elevated during early cancerogenesis.Steroids. 2001; 66: 287-292Crossref PubMed Scopus (186) Google Scholar, 23.Yano S. Sugimoto T. Tsukamoto T. et al.Decrease in vitamin D receptor and calcium-sensing receptor in highly proliferative parathyroid adenomas.Eur J Endocrinol. 2003; 148: 403-411Crossref PubMed Scopus (72) Google Scholar However, increases in VDR could also stimulate the expression of 24-hydroxylase, the enzyme that initiates the degradation of 1,25(OH)2D3, in order to regulate its local concentrations and, therefore, the response to 1,25(OH)2D3. In addition, in this work, we have found that 1,25(OH)2D3 has a clear proliferative effect. As the first report stating that 1,25(OH)2D3 inhibited proliferation and induced the differentiation of murine myeloid leukemia M1 cells into monocyte macrophages,24.Miyaura C. Abe E. Suda T. Extracellular calcium is involved in the mechanism of differentiation of mouse myeloid-leukemia cells (M1) induced by 1-alpha,25-dihydroxyvitamin-D3.Endocrinology. 1984; 115: 1891-1896Crossref PubMed Scopus (15) Google Scholar many studies have demonstrated that 1,25(OH)2D3 is a potent suppressor of proliferation and an inductor of the differentiation in numerous cells types.25.Weinreich T. Muller A. Wuthrich R.P. et al.1,25-Dihydroxyvitamin D-3 and the synthetic: vitamin D analogue, KH 1060, modulate the growth of mouse proximal tubular cells.Kidney Blood Pressure Res. 1996; 19: 325-331Crossref PubMed Scopus (17) Google Scholar, 26.Ylikomi T. Laaksi I. Lou Y.R. et al.Antiproliferative action of vitamin D.Vitamins Hormones – Adv Res Appl. 2002; 64: 357-406Crossref PubMed Google Scholar However, the few studies aimed at analyzing the effect of 1,25(OH)2D3 on VSMC proliferation showed conflictive results, most likely owing to the different experimental conditions used.18.Maccarthy E.P. Yamashita W. Hsu A. et al.1,25-Dihydroxyvitamin-D3 and rat vascular smooth-muscle cell-growth.Hypertension. 1989; 13: 954-959Crossref PubMed Google Scholar, 27.Mitsuhashi T. Morris R.C. Ives H.E. 1,25-Dihydroxyvitamin-D3 modulates growth of vascular smooth-muscle cells.J Clin Invest. 1991; 87: 1889-1895Crossref PubMed Scopus (163) Google Scholar Furthermore, the discovery of the VDR in VSMC raises new questions about its function in this particular cell type. We have used quiescent cells to prevent saturating effects of other growth factors. Furthermore, no serum was added in the experiment. This is particularly important because it demonstrates that 1,25(OH)2D3 alone, in the absence of any other stimuli, is able to induce an increase in VSMC proliferation. Thus, the effect of 1,25(OH)2D3 on VSMC proliferation is direct, and independent of serum-related factors. In a second approach, the effect of 1,25(OH)2D3 on the proliferation of VSMC already stimulated to grow was assessed. We found that the effects of 1,25(OH)2D3 were additive to those of bFGF, demonstrating that the effect of 1,25(OH)2D3 on VSMC proliferation is not dependent on the status of the cell (quiescent vs not quiescent). Thus, in pathological conditions in which VSMCs are already stimulated to grow, the addition of 1,25(OH)2D3 could lead even to a higher increase of the proliferation rate. This could be especially important in patients with CKD, in which treatment with 1,25(OH)2D3 is a common medical practice. It is known that patients with CKD have an increase in VSMC proliferation, leading to arterial complications and uremic arteriopathy.6.Amann K. Wolf B. Nichols C. et al.Aortic changes in experimental renal failure – hyperplasia or hypertrophy of smooth muscle cells?.Hypertension. 1997; 29: 770-775Crossref PubMed Scopus (92) Google Scholar Therefore, treatment with 1,25(OH)2D3 could lead to a further stimulus in VSMC proliferation. In a third approach, we added crescent amounts of serum to the cells stimulated with 1,25(OH)2D3. In this case, 1,25(OH)2D3 was not able to increase further proliferation rates when cells were strongly stimulated to proliferate by high concentrations of serum. However, 1,25(OH)2D3 was able to increase the proliferation rate in cells incubated with lower levels of serum. This could be explained by the fact that cells incubated in vitro have a differential uptake of 1,25(OH)2D3 compared to that of VSMCs surrounded by an intact arterial structure. A further evidence of the increase in proliferation induced by 1,25(OH)2D3 was obtained by fluorescence-activated cell sorter analysis. Our results indicate that VSMCs enter S phase earlier in the presence of 1,25(OH)2D3, suggesting that the higher proliferation rates caused by 1,25(OH)2D3 are probably owing to a shortening of the G1 phase. These in vitro results must be taken with caution, because of the absence of inhibitory factors present in the in vivo situation, when other cell types are present. However, the proliferative effect of 1,25(OH)2D3 has also been demonstrated ex vivo in arterial rings and in vivo in animals with CKD. This in vivo effect could also be an indirect effect mediated by the increase in serum calcium and phosphate levels. Nevertheless, we must point out that the doses uses in vivo in our experimental settings are higher than the ones used in patients, reflecting the effect of 1,25(OH)2D3 overdosing. Extrapolating this effect to the clinical field, 1,25(OH)2D3 might be an important mediator of arterial remodelling induced by atherogenic factors, direct injury, uremia, and hemodynamic burden. Furthermore, the fact that VDR is upregulated by 1,25(OH)2D3 in VSMC raises new concerns. Thus, if 1,25(OH)2D3 is overdosed in chronic renal failure28.Fernandez E. Llach F. Guidelines for dosing of intravenous calcitriol in dialysis patients with hyperparathyroidism.Nephrol Dial Transplant. 1996; 11: 96-101Crossref PubMed Scopus (24) Google Scholar or osteoporosis,29.Kitchin B. Morgan S. Nutritional considerations in osteoporosis.Curr Opin Rheumatol. 2003; 15: 476-480Crossref PubMed Scopus (25) Google Scholar the beneficial effects of the therapy might be shadowed by an increase of the susceptibility of vessels to 1,25(OH)2D3-induced proliferation. The concentration of 1,25(OH)2D3 used in this study (5–100 nM) is higher than the normal serum levels, but it falls within the range which induces biological response in other tissues and cell types analyzed in vitro.18.Maccarthy E.P. Yamashita W. Hsu A. et al.1,25-Dihydroxyvitamin-D3 and rat vascular smooth-muscle cell-growth.Hypertension. 1989; 13: 954-959Crossref PubMed Google Scholar, 30.Brown A.J. Ritter C. Slatopolsky E. et al.1 Alpha,25-dihydroxy-3-epi-vitamin D-3, a natural metabolite of 1 alpha,25-dihydroxyvitamin D-3, is a potent suppressor of parathyroid hormone secretion.J Cell Biochem. 1999; 73: 106-113Crossref PubMed Scopus (99) Google Scholar, 31.Canalejo A. Almaden Y. Torregrosa V. et al.The in vitro effect of calcitriol on parathyroid cell proliferation and apoptosis.J Am Soc Nephrol. 2000; 11: 1865-1872PubMed Google Scholar, 32.Cordero J.B. Cozzolino M. Lu Y. et al.1,25-Dihydroxyvitamin D down-regulates cell membrane growth- and nuclear growth-promoting signals by the epidermal growth factor receptor.J Biol Chem. 2002; 277: 38965-38971Crossref PubMed Scopus (98) Google Scholar, 33.Finch J.L. Dusso A.S. Pavlopoulos T. et al.Relative potencies of 1,25-(OH)(2)D-3 and 19-Nor-1,25-(OH)(2)D-2 on inducing differentiation and markers of bone formation in MG-63 cells.J Am Soc Nephrol. 2001; 12: 1468-1474PubMed Google Scholar Furthermore, in the clinical settings, the levels of 25-hydroxyvitamin D are used to determine the vitamin D status. The normal range of 25-hydroxyvitamin D-circulating levels is 40–180 nM. Thus, knowing that endothelial cells possess the capacity to transform 25-hydroxyvitamin D in 1,25(OH)2D3 and that this activity seems to be only related to substrate availability,16.Zehnder D. Bland R. Chana R.S. et al.Synthesis of 1,25-dihydroxyvitamin D-3 by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion.J Am Soc Nephrol. 2002; 13: 621-629Crossref PubMed Google Scholar we can suggest that local levels at the vascular bed could be much higher than in serum. Furthermore, in renal failure, extrarenal synthesis and degradation of 1,25(OH)2D3 seems to be altered. Dusso et al.34.Dusso A.S. Finch J. Brown A. et al.Extrarenal production of calcitriol in normal and uremic humans.J Clin Endocrinol Metab. 1991; 72: 157-164Crossref PubMed Scopus (110) Google Scholar demonstrated that macrophages showed an increased synthesis of 1,25(OH)2D3 in uremic conditions. Gallieni et al.35.Gallieni M. Kamimura S. Ahmed A. et al.Kinetics of monocyte 1-alpha-hydroxylase in renal-failure.Am J Physiol – Renal Physiol. 1995; 37: F746-F753Google Scholar showed that monocytes also had an increase 1-α hydroxylase activity in uremia. In addition, Hsu et al.36.Hsu C.H. Patel S.R. Young E.W. Mechanism of decreased calcitriol degradation in renal-failure.Am J Physiol. 1992; 262: F192-F198PubMed Google Scholar studied the effect of uremic toxins on 24 and 25 hydroxylase activities. This study concluded that, in uremic conditions, metabolic degradation of 1,25(OH)2D3 is decreased. There is no direct evidence that the synthesis of 1,25(OH)2D3 by endothelial cells is increased in renal failure. However, it has been published that the synthesis of 1,25(OH)2D3 by human endothelial cells is increased by inflammatory cytokines16.Zehnder D. Bland R. Chana R.S. et al.Synthesis of 1,25-dihydroxyvitamin D-3 by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion.J Am Soc Nephrol. 2002; 13: 621-629Crossref PubMed Google Scholar and that in renal failure there is a systemic inflammatory condition with an increase in tumor necrosis factor-α and interleukin-8.16.Zehnder D. Bland R. Chana R.S. et al.Synthesis of 1,25-dihydroxyvitamin D-3 by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion.J Am Soc Nephrol. 2002; 13: 621-629Crossref PubMed Google Scholar, 37.Mezzano D. Pais E.O. Aranda E. et al.Inflammation, not hyperhomocysteinemia, is related to oxidative stress and hemostatic and endothelial dysfunction in uremia.Kidney Int. 2001; 60: 1844-1850Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar In addition, advanced glycation end products, which accumulate in renal failure, increase tumor necrosis factor-α expression in endothelial cells.16.Zehnder D. Bland R. Chana R.S. et al.Synthesis of 1,25-dihydroxyvitamin D-3 by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion.J Am Soc Nephrol. 2002; 13: 621-629Crossref PubMed Google Scholar, 38.Rashid G. Benchetrit S. Fishman D. et al.Effect of advanced glycation end-products on gene expression and synthesis of TNF-alpha and endothelial nitric oxide synthase by endothelial cells.Kidney Int. 2004; 66: 1099-1106Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Thus, in renal failure conditions, the endothelial synthesis of 1,25(OH)2D3 could be increased, leading to high local levels. There is now a consensus that VEGF is crucial in pathological processes both in embryo and in adults. Alterations of VEGF expression have been involved in several pathologies, like tumor growth,39.Ferrara N. VEGF and the quest for tumour angiogenesis factors.Nat Rev Cancer. 2002; 2: 795-803Crossref PubMed Scopus (1198) Google Scholar pre-eclampsia,40.Maynard S.E. Min J.Y. Merchan J. et al.Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Crossref PubMed Scopus (2875) Google Scholar etc. Furthermore, a recent paper stated the involvement of VEGF in neointimal proliferation in pig arteries following stent implantation.41.Shibata M. Suzuki H. Nakatani M. et al.The involvement of vascular endothelial growth factor and flt-1 in the process of neointimal proliferation in pig coronary arteries following stent implantation.Histochem Cell Biol. 2001; 116: 471-481Crossref PubMed Scopus (56) Google Scholar Also, Parenti et al.42.Parenti A. Bellik L. Brogelli L. et al.Endogenous VEGF-A is responsible for mitogenic effects of MCP-1 on vascular smooth muscle cells.Am J Physiol – Heart Circ Physiol. 2004; 286: H1978-H1984Crossref PubMed Scopus (56) Google Scholar demonstrated that VSMC proliferation induced by monocyte chemoattractant protein-1 was mediated by endogenous production of VEGF-A. Taken together, these results show that VSMC can both produce and respond to VEGF. Our results also indicate that 1,25(OH)2D3 induces an increase in the expression of VEGF. These results are in agreement with those of Yamamoto et al.,43.Yamamoto T. Kozawa O. Tanabe K. et al.1,25-Dihydroxyvitamin D-3 stimulates vascular endothelial growth factor release in aortic smooth muscle cells: role of p38 mitogen-activated protein kinase.Arch Biochem Biophys. 2002; 398: 1-6Crossref PubMed Scopus (49) Google Scholar which showed an increase in VEGF release from a stable line of smooth muscle cells in culture. Furthermore, VSMC express VEGF receptors,44.Ishida A. Murray J. Saito Y. et al.Expression of vascular endothelial growth factor receptors in smooth muscle cells.J Cell Physiol. 2001; 188: 359-368Crossref PubMed Scopus (176) Google Scholar making them a possible target for the effects of VEGF. We also found that the addition of the VEGF receptor-binding antagonist VGA110245.Ueda Y. Yamagishi T. Samata K. et al.A novel low molecular weight VEGF receptor-binding antagonist VGA1102, inhibits the function of VEGF and in vivo tumor growth.Cancer Chemother Pharmacol. 2004; 54: 16-24Crossref PubMed Scopus (12) Google Scholar or VEGF-neutralizing antibodies blunted the increase in 5-bromo-2′-deoxy-uridine (BrdU) incorporation levels in 1,25(OH)2D3-stimulated cells, suggesting that 1,25(OH)2D3-induced VSMC proliferation is mediated by VEGF. The mechanism by which 1,25(OH)2D3 increases VEGF production in VSMC is not clear, but the presence of a sequence that resembles the DR3 type of vitamin D-responsive elements46.Haussler M.R. Whitfield G.K. Haussler C.A. et al.The nuclear vitamin D receptor: biological and molecular regulatory properties revealed.J Bone Miner Res. 1998; 13: 325-349Crossref PubMed Scopus (1167) Google Scholar in the promoter of the rat VEGF gene may provide an explanation. Further experiments are needed to determine whether this sequence binds VDR and enhances transcription in a vitamin D-dependent m
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