J. Ruth Wu-Wong

Vitamin D Analogs Modulate the Expression of Plasminogen Activator Inhibitor-1, Thrombospondin-1 and Thrombomodulin in Human Aortic Smooth Muscle Cells

Background/Aims: Plasminogen activator inhibitor-1 (PAI-1), thrombospondin-1 (THBS1) and thrombomodulin (TM) are involved in atherothrombosis. Vitamin D receptor agonists (VDRAs) provide survival/cardiovascular benefits for chronic kidney disease patients. Methods: The effects of VDRAs on regulating PAI-1, THBS1 and TM in human aortic smooth muscle cells (SMC) and endothelial cells (EC) were studied. Results: In SMC, paricalcitol and calcitriol downregulated the expression of PAI-1 mRNA and protein in a dose-dependent manner (EC50 = 0.7 and 4.4 nM, respectively). Both drugs also downregulated THBS1 mRNA and protein (EC50 = 1.6 and 3.9 nM, respectively). In contrast, paricalcitol and calcitriol upregulated TM mRNA and protein (EC50 = 28.9 and 25.5 nM, respectively). EC did not express VDR, and VDRAs failed to induce CYP24A1, a VDR target gene. The effect of paricalcitol on THBS1 in SMC was blocked by cycloheximide, while its effect on TM and CYP24A1 was not affected, suggesting that the regulation of THBS1 by VDR may be mediated through intermediate factors, but that TM is likely a direct target of VDR. Conclusion: VDR may play a role in atherothrombosis via regulation of PAI-1, THBS1 and TM.

Pharmacology of endothelin receptor antagonists ABT-627, ABT-546, A-182086 and A-192621: ex vivo and in vivo studies.

Endothelins (ETs), 21-amino-acid peptides involved in the pathogenesis of various diseases, bind to ET(A) and ET(B) receptors to initiate their effects. Based on the same core structure, we have developed four small-molecule ET receptor antagonists, ABT-627 (atrasentan), ABT-546, A-182086 and A-192621, which exhibit differences in selectivity for ET(A) and ET(B) receptors. In this report, we compare the efficacy, potency and pharmacokinetic properties of these four antagonists, including potency in inhibiting ET-1- or Sarafotoxin 6c-induced vessel constriction in isolated arteries and efficacy in antagonizing ET-1-, big ET-1- or Sarafotoxin 6c-induced pressor responses in rats.

Differential Inhibition of Renin mRNA Expression by Paricalcitol and Calcitriol in C57/BL6 Mice

Background/Aims: Vitamin D receptor activators (VDRAs) may suppress renin expression and VDR-mediated renin inhibitors may offer a novel mechanism to control the RAS. Methods: We delineated the effects of paricalcitol and calcitriol on PTH, renin, and iCa2+ in C57/BL6 mice administered vehicle, paricalcitol, or calcitriol (0.01, 0.03, 0.10, 0.33, 1.0 µg/kg s.c.) 3 days/week for 9 days. Results: Paricalcitol produced PTH suppression from 0.03 to 1.0 µg/kg (values between 9.7 ± 3.3 and 20.7 ± 4.7 pg/ml; vehicle = 88.0 ± 16.9) and elicited dose-dependent reductions in renin/GAPDH expression at 0.33 and 1.0 µg/kg (0.037 ± 0.002, 0.027 ± 0.003; vehicle = 0.054 ± 0.003) but produced no increases iCa2+ at any dose tested. Calcitrol produced PTH suppression at all doses tested (between 6.4 ± 1.2 and 29.5 ± 17.2 pg/ml) and renin suppression at 0.10, 0.33, and 1.0 µg/kg (0.029 ± 0.002, 0.031 ± 0.003, and 0.038 ± 0.02). However, at 0.33 and 1.0 mg/kg, calcitriol produced increases iCa2+ (1.31 ± 0.03 and 1.48 ± 0.02 mmol/l; vehicle = 1.23 ± 0.02 mmol/l). Conclusions: Paricalcitol produces significant, dose-dependent suppression of renin expression in the absence of hypercalcemia at doses 10-fold above those necessary for PTH suppression. Calcitriol also produced suppression of renin at doses at least 10-fold above those required for PTH suppression, but increases in iCa2+ were observed at doses only 3-fold above those necessary to elicit renin suppression.

Vitamin D Receptor Activation Mitigates the Impact of Uremia on Endothelial Function in the 5/6 Nephrectomized Rats

Endothelial dysfunction increases cardiovascular disease risk in chronic kidney disease (CKD). This study investigates whether VDR activation affects endothelial function in CKD. The 5/6 nephrectomized (NX) rats with experimental chronic renal insufficiency were treated with or without paricalcitol, a VDR activator. Thoracic aortic rings were precontracted with phenylephrine and then treated with acetylcholine or sodium nitroprusside. Uremia significantly affected aortic relaxation (−50.0 ± 7.4% in NX rats versus −96.2 ± 5.3% in SHAM at 30 μM acetylcholine). The endothelial-dependent relaxation was improved to –58.2 ± 6.0%, –77.5 ± 7.3%, and –90.5 ± 4.0% in NX rats treated with paricalcitol at 0.021, 0.042, and 0.083 μg/kg for two weeks, respectively, while paricalcitol at 0.042 μg/kg did not affect blood pressure and heart rate. Parathyroid hormone (PTH) suppression alone did not improve endothelial function since cinacalcet suppressed PTH without affecting endothelial-dependent vasorelaxation. N-omega-nitro-L-arginine methyl ester completely abolished the effect of paricalcitol on improving endothelial function. These results demonstrate that VDR activation improves endothelial function in CKD.

Pharmacology of endothelin receptor antagonists ABT-627, ABT-546, A-182086 and A-192621: in vitro studies.

Endothelins (ETs), 21-amino-acid peptides involved in the pathogenesis of various diseases, bind to ET(A) and ET(B) receptors to initiate their effects. Based on the same core structure, we have developed four small-molecule ET receptor antagonists, ABT-627, ABT-546, A-182086 and A-192621, which exhibit difference in selectivity for ET(A) and ET(B) receptors. In this report, we compare the potency and selectivity of these four antagonists in inhibiting (125)I-labelled ET-1 binding to cloned human ET(A) and ET(B) receptors, and in blocking ET-1-induced functional responses (arachidonic acid release and phosphatidylinositol hydrolysis).

Differential effects of Vitamin D analogs on bone formation and resorption

Deficiency in Vitamin D and its metabolites leads to a failure in bone formation primarily caused by dysfunctional mineralization, suggesting that Vitamin D analogs might stimulate osteoblastic bone formation and mineralization. In this study, we compare the effect of selected Vitamin D analogs and active metabolite, 1alpha,25-dihydroxyvitamin D(3), 19-nor-1alpha, 25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) or 1alpha,25-dihydroxyvitamin D(2) on bone formation and resorption. In a mouse calvariae bone primary organ culture system, all Vitamin D analogs and metabolite tested-stimulated collagen synthesis in a dose-dependent manner and 19-nor-1alpha, 25-dihydroxyvitamin D(2) was the most efficacious among three. 19-nor-1alpha, 25-dihydroxyvitamin D(2) and 1alpha,25-dihydroxyvitamin D(2) showed similar potencies and 1alpha,25-dihydroxyvitamin D(3) was less potent than others. Osteocalcin was also up-regulated in a dose-dependent manner, suggesting that the three Vitamin D analogs have the equal potencies on bone formation. 25-Hydroxyvitamin D-24-hydroxylase expression was induced in a dose-dependent manner and 19-nor-1alpha, 25-dihydroxyvitamin D(2) was less potent than other two compounds. In a mouse calvariae organ culture, all induced a net calcium release from calvariae in a dose-dependent manner, but the potency is in the order of 1alpha,25-dihydroxyvitamin D(2) congruent with1alpha,25-dihydroxyvitamin D(3)>19-nor-1alpha, 25-dihydroxyvitamin D(2). In a Vitamin D/calcium-restricted rat model, all caused an elevation in serum calcium in a dose-dependent manner. There is no significant difference between 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(2) in potencies, but 19-nor-1alpha, 25-dihydroxyvitamin D(2) is at least 10-fold less potent than the other two compounds. Our results suggest that Vitamin D analogs have direct effects on bone resorption and formation, and 19-nor-1alpha, 25-dihydroxyvitamin D(2) may be more effective than 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(2) on stimulating anabolic bone formation.

Systemic Activation of the Calcium Sensing Receptor Produces Acute Effects on Vascular Tone and Circulatory Function in Uremic and Normal Rats: Focus on Central versus Peripheral Control of Vascular Tone and Blood Pressure by Cinacalcet

Calcium-sensing receptor (CaR) activation decreases serum parathyroid hormone (PTH) and Ca2+ and, despite long-term reductions in mean arterial blood pressure (MAP), may produce acute hypertension in rats, an effect we hypothesized was mediated by constriction of multiple vascular beds. Rats were subjected to 5/6 nephrectomy (NX) or no surgery (Normal); at 7 to 8 weeks, uremia animals were anesthetized and instrumented to record MAP and regional blood flow (carotid, mesenteric, and hindlimb). Cinacalcet [N-(1-naphthalen-1-ylethyl)-3-[3-(trifluoromethyl)phenyl]-propan-1-amine; 1, 3, and 10 mg/kg; 30 min/dose] was infused over 90 min. In NX rats, cinacalcet dose-dependently decreased ionized calcium (iCa2+), elicited a 90% reduction in PTH, and produced dose-dependent self-limiting increases in MAP (from 119 ± 6to129 ± 5, 142 ± 4, and 145 ± 3 mm Hg at the end of each infusion). At 1 mg/kg, carotid vascular resistance (CVR) and mesenteric vascular resistance (MVR) increased to 16 ± 6 and 18 ± 6% above baseline, respectively. Hindlimb vascular resistance (HVR) also trended upward (13 ± 8%). At 3 mg/kg, increases in CVR (38 ± 10%), MVR (40 ± 8%), and HVR (39 ± 14%) were exacerbated; at 10 mg/kg, values remained at or near these levels. The effects of cinacalcet in Normal rats were similar to NX and were attenuated by ganglionic blockade with hexamethonium at low doses but remained significantly elevated at higher doses. Thus, CaR activation acutely increases MAP in uremic and nonuremic rats, responses that occur in parallel to vasoconstriction in multiple vascular beds through both a central and peripheral mechanism of action. Moreover, subsequent mechanistic studies suggest that increases in MAP produced by cinacalcet may be mediated by reduced tonic NO synthase-dependent NO production subsequent to reductions in blood iCa2+.

Differential effects of Vitamin D analogs on calcium transport.

It is well known that the efficiency of intestinal active calcium transport is regulated by the Vitamin D receptor pathway and Vitamin D analogs seem to exhibit differential effects on intestinal active calcium transport. To investigate the molecular basis for the difference among Vitamin D analogs, we tested three Vitamin D analogs: 1,25-dihydroxyvitamin D(3), 19-nor-1,25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) ex vivo and in vitro. In 5/6 nephrectomized rat intestinal active calcium transport, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effects on intestinal active calcium transport at all the concentrations tested, while 1alpha-hydroxyvitamin D(2) at 0.33 and 0.67 microg/kg and 1,25-dihydroxyvitamin D(3) at 1microg/kg significantly stimulated calcium transport. In Caco-2 cells, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effect on calcium transport, while 1,25-dihydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(2) (the active form of 1alpha-hydroxyvitamin D(2)) stimulated calcium transport by 934 and 501% at 0.1microM, respectively. 1,25-Dihydroxyvitamin D(2) potently induced the expression of CALB3 and TRPV6 mRNA with an EC(50) of 0.3 and 1.0nM, whereas 19-nor-1,25-dihydroxyvitamin D(2) was 10-fold less potent than 1,25-dihydroxyvitamin D(2) in inducing CALB3 and TRPV6 mRNA. The three Vitamin D analogs had no significant effect on the expression of PMCA1 mRNA. These Vitamin D analogs did not change the expression of Vitamin D receptor (VDR) up to 10nM, but stimulated CYP24A1 expression in a dose-dependent manner with the potency in the order of 1,25-dihydroxyvitamin D(3)>1,25-dihydroxyvitamin D(2)=19-nor-1,25-dihydroxyvitamin D(2). These results suggest that the differential effect of Vitamin D analogs on stimulating intestinal and Caco-2 calcium transport may be in part due to its different effect on stimulating CALB3 and TRPV6 mRNA expression.

VS‐105: a novel vitamin D receptor modulator with cardiovascular protective effects

BACKGROUND AND PURPOSE Vitamin D receptor (VDR) modulators (VDRMs) such as calcitriol, paricalcitol and doxercalciferol are commonly used to manage hyperparathyroidism secondary to chronic kidney disease (CKD). CKD patients experience extremely high risks of cardiovascular morbidity and mortality. Clinical observations show that VDRM therapy may be associated with cardio‐renal protective and survival benefits for CKD patients. However, hypercalcaemia remains a serious side effect for current VDRMs, which leads to the need for frequent dose titration and serum Ca (calcium) monitoring. Significant clinical benefits can be derived from a VDRM with cardiovascular protective effects without the hypercalcaemic liability.

Effect of castration on endothelin receptors

Endothelin (ET) plays a pivotal role in the pathogenesis of cell growth disorders such as cancer. Atrasentan (ABT-627), a selective antagonist for the ET receptor A (ET(A)), has shown benefit in controlling disease progression in men with hormone refractory prostate cancer who have undergone aggressive hormone ablation therapy. It is not known how hormone ablation affects ET-binding sites, although ET-1 and ET(A) expression are found to be elevated in prostate cancer patients. In this study, we examined the effect of castration on ET receptor binding in male beagle dogs. Three dogs were surgically castrated and another three sham-operated. The animals were sacrificed 1 week after operation and membranes were prepared from the prostate, heart, brain, kidney, liver and lung for ET-1, ET-3 and angiotensin II (A-II, as a control) binding studies. No significant difference in A-II binding was observed between castrated and sham-operated animals. However, ET-1 and ET-3 binding to prostate and brain membranes were altered significantly. From saturation binding studies using ET-1 in the prostate, the K(d) and B(max) values increased from 0.043 nM and 0.094 pmol/mg respectively in sham-operated dogs to 0.104 nM and 0.311 pmol/mg respectively in castrated animals. These results indicate that surgical castration in dogs produces a change in the ET receptor density in the prostate and brain, and may have implications for the effect of hormone ablation therapy on ET receptor expression in prostate cancer patients.