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Endothelial cell migration and proliferation are the key steps in the angiogenic process, and both are stimulated by recombinant human erythropoietin (rHuEPO). In addition rHuEPO can increase endothelin-1 (ET-1) release by the endothelial cell. We designed the present study to address the question of whether rHuEPO stimulates angiogenesis. An in vitro quantitative assay for angiogenesis was used. This consisted of rat aortic rings embedded in a reconstituted basement membrane matrix and incubated with and without rHuEPO for eight days. We found that rHuEPO increased vessel outgrowth after four days of culture and this was continued for the next four days (rHuEPO vs. control: day 4, 12 +/- 2 vs. 4 +/- 1, P < 0.002 and day 8, 124 +/- 18 vs. 56 +/- 12 P < 0.006). Supernatant endothelin-1 (ET-1) levels, at 24 hours, were significantly higher than controls in the rings incubated with rHuEPO (107 +/- 13 vs. 43 +/- 10 pg/ml, P < 0.003). To investigate the role of ET-1 in rHuEPO-induced angiogenesis, rings were exposed to ET-1 alone (10(-8) M). We observed an increase in microvessel formation compared to control (day 4, 4 +/- 2 vs. 2 +/- 1, P < 0.006, and day 8, 67 +/- 12 vs. 51 +/- 10, P < 0.03). In addition, aortic rings were co-cultured with rHuEPO and anti-ET-1 IgG antibody. Stimulation of angiogenesis by rHuEPO was blunted by the ET-1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypertension is a major complication of rHuEPO therapy in hemodialysis (HD) patients. We have previously reported that patients receiving rHuEPO intravenously (i.v.) had higher mean arterial pressure (MAP) and plasma endothelin-1 (ET-1) levels than those in which the hormone was administered subcutaneously (s.c.). To test whether the increased serum ET-1 levels associated with i.v. rHuEPO administration are the result of a direct effect of the hormone on ET-1 release by the endothelial cells (EC), we examined the effects of rHuEPO in vitro. Bovine pulmonary artery endothelial cells (BPAEC) were exposed to doses of rHuEPO of 0.8; 1.6; 3.3 and 6.6 U/ml. A 24 hour-time course showed maximal ET-1 production at 12 hours for all the doses tested. A significant increase in cell proliferation over controls was observed at 24 hours, for all rHuEPO doses, and no correlation was found between ET-1 values and cell proliferation. Inhibition of protein synthesis by cycloheximide (10 micrograms/ml) abolished the stimulation of ET-1 release by rHuEPO. Thrombin (4 U/ml) and angiotensin II (10(-7) M), two potent stimulators of ET-1 release, had additive effects to those of rHuEPO. Specific thrombin and angiotensin II antagonists blocked these additive effects, reducing ET-1 release to the level of rHuEPO stimulation alone. In summary, rHuEPO stimulates vascular EC in culture to increase ET-1 release through an increase in synthesis and in a time dependent fashion. The routes of stimulation seem to differ from other known ET-1 secretogoges. Our data also confirm a significant mitogenic effect of rHuEPO on the endothelial cell.

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Idiopathic hypercalciuria (IH) is a heterogeneous disorder frequently observed in patients with nephrolithiasis. At one extreme of its clinical spectrum is fasting hypercalciuria (FH), a condition characterized by increased bone resorption and turnover. In previous studies we have shown that monocytes from patients with high turnover osteoporosis and from women in early postmenopause elaborate increased amounts of interleukin-1 (IL-1), a cytokine that stimulates bone resorption in vitro and in vivo. Since IL-1 could also mediate the resorptive mechanism of FH and cause a clinically significant bone loss, we have studied the relationship of IH, vertebral mineral density, bone turnover, and monocyte IL-1 activity in 47 patients with absorptive hypercalciuria (AH), 23 with FH, and 38 nonhypercalciuric subjects with recurrent nephrolithiasis (controls). Vertebral mineral density, as measured by quantitative computer tomography, was decreased in each of the three patient groups, but was significantly lower in FH patients than in AH patients or control subjects. Twenty-four-hour total urinary hydroxyproline excretion was increased in FH patients compared to that in AH patients or controls, but blood levels of osteocalcin were not. Monocytes from FH subjects yielded significantly more IL-1 (alpha + beta) activity than those from AH patients or controls; levels of IL-1 activity in monocytes of AH and control patients were similar. In IH subjects, significant correlations were found between IL-1 and hydroxyproline (r = 0.70; P less than 0.0001), IL-1 and quantitative computer tomography values (r = -0.49; P less than 0.005), and IL-1 and urinary calcium (r = -0.36; P less than 0.05). Serum PTH levels were within normal limits in all subjects and were similar in the three study groups, 1,25-Dihydroxyvitamin D3 levels, although higher in IH patients than in controls, were not significantly different in FH and AH subjects. Increased IL-1 activity and decreased vertebral mineral density are features of a subset of patients with IH. Although a cause-effect relationship remains to be established, increased monocytic IL-1 activity, rather than elevated PTH or 1,25-dihydroxyvitamin D3 levels, could underlie the resorptive component of FH.

Background: The phosphatonins fibroblast growth factor-23 (FGF-23) and FRP-4 are inhibitors of tubular phosphate reabsorption that may play a role in the hyperphosphatemia associated with chronic kidney disease (CKD) or in the hypophosphatemia associated with renal transplants. Methods: Plasma FGF-23, FRP-4, phosphorus and parathyroid hormone were measured in patients at all stages of CKD. Phosphate regulation of FGF-23 and secreted frizzled related protein-4 (sFRP-4) was examined in end-stage renal disease patients in the presence and absence of therapeutic phosphate binder usage. In renal transplant patients, plasma FGF-23, sFRP-4 and phosphorus concentrations were determined before and 4–5 days after transplantation. Results: Plasma FGF-23 correlated with creatinine clearance (r² = –0.584, p < 0.0001) and plasma phosphorus (r² = 0.347, p < 0.001) in CKD patients and with plasma phosphorus (r² = 0.448, p < 0.001) in end-stage renal disease patients. Phosphate binder withdrawal increased FGF-23 levels. In kidney transplant patients, dramatic decreases in FGF-23 (–88.8 ± 5.4%) and phosphorus (–64 ± 10.2%) were observed by 4–5 days post-transplantation. In patients with post-transplant hypophosphatemia, FGF-23 levels correlated inversely with plasma phosphorus (r² = 0.661, p < 0.05). sFRP-4 levels did not change with creatinine clearance or hyperphosphatemia in CKD or end-stage renal disease patients, and no relation was noted between post-transplant sFRP-4 levels and hypophosphatemia. Conclusions: In CKD, FGF-23 levels rose with decreasing creatinine clearance rates and increasing plasma phosphorus levels, and rapidly decreased post-transplantation suggesting FGF-23 is cleared by the kidney. Residual FGF-23 may contribute to the hypophosphatemia in post-transplant patients.

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These results suggest that rHuEPO prevents LPS-induced apoptosis in endothelial cells. This protective effect could be an important factor in the action of rHuEPO on vascular endothelium.