Junzheng Peng

Enzymatic Activity of Lysosomal Carboxypeptidase (Cathepsin) A Is Required for Proper Elastic Fiber Formation and Inactivation of Endothelin-1

Background— Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomal multienzyme complex along with &bgr;-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GAL and Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactive splice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA cause galactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomal storage of sialylated glycoconjugates. However, several phenotypic features of galactosialidosis patients, including hypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest that CathA may be involved in hemodynamic functions that go beyond its protective activity in the lysosome. Methods and Results— We generated a gene-targeted mouse in which the active CathA was replaced with a mutant enzyme carrying a Ser190Ala substitution in the active site. These animals expressed physiological amounts of catalytically inactive CathA protein, capable of forming lysosomal multienzyme complex, and did not develop secondary deficiency of Neu1 and GAL. Conversely, the mice showed a reduced degradation rate of the vasoconstrictor peptide, endothelin-1, and significantly increased arterial blood pressure. CathA-deficient mice also displayed scarcity of elastic fibers in lungs, aortic adventitia, and skin. Conclusions— Our results provide the first evidence that CathA acts in vivo as an endothelin-1–inactivating enzyme and strongly confirm a crucial role of this enzyme in effective elastic fiber formation.

Effect of tacrolimus (FK506) and sirolimus (rapamycin) mono- and combination therapy in prolongation of renal allograft survival in the monkey

BACKGROUND Our previous studies confirmed that tacrolimus (FK506) and sirolimus [rapamycin (RAPA)], in combination, are not antagonistic but are synergistic in the prolongation of heart and small bowel grafts in the rodent. The aim of this study was to confirm further the synergistic effect of combined FK506 and RAPA in the more clinically relevant model, kidney transplantation in monkeys. METHODS A total of 60 male Vervet monkeys were randomly assigned to 10 groups (n> or =5). Monkeys with renal allografts were treated with different doses of FK506 and/or RAPA orally for 60 days. Graft survival, body weight, clinical biochemistry determinations, oral glucose tolerance test, trough levels of the two drugs, and histopathology were investigated. RESULTS Low doses of FK506 (1 or 4 mg/kg) combined with RAPA (0.5 mg/kg) produced synergistic effect in the prolongation of renal graft survival [combination index (CI) = 0.292, 0.565]. There were no additive or synergistic drug-associated toxicities such as hyperglycemia, nephrotoxicity, and hyperlipidemia. There also was no pharmacological antagonism. CONCLUSION Concomitant therapy of low-dose (drug-optimal) FK506 and RAPA produced a synergistic effect in the prolongation of kidney allograft survival in Vervet monkeys without additive drug-associated toxicities.

Synergistic effects of mycophenolate mofetil and sirolimus in prevention of acute heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat

BACKGROUND The effect of mycophenolate mofetil (MMF) and sirolimus (rapamycin, RAPA) mono- and combination-therapy was examined in prevention of acute heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat. METHODS Both drugs were administered orally for up to 30 days. Eleven groups (n=6) were involved in the first part of the heart allografting model. Brown Norway (RT1n) to Lewis (RT1(1)) combination was used in the heart and pancreas transplantation models, whereas Buffalo (RT1b) to Wistar Furth (RT1u) was used in the kidney transplantation model. RESULTS The naive control group showed a mean survival time of 6.5+/-0.6 days. There were graded dose-responses to monotherapy of MMF 10 and 20 mg(kg/ day (12.5+/-2.6 days; 19.3+/-9.0 days) and RAPA 0.2, 0.4, 0.8, and 1.8 mg/kg/day (19.2+/-2.0 days; 30.0+/-7.3 days; 50.8+/-12.5 days; 51.2+/-2.6 days), respectively (P=0.001). Results with the combined use of drugs indicate that a synergistic or very strong synergistic interaction was produced when compared with monotherapy of MMF or RAPA: MMF 10 mg(kg/day+RAPA 0.2 mg/kg(day (52.7+/-5.7 days, combination index [CI] =0.189), MMF 20 mg(kg/day+RAPA 0.2 mg/kg/day (57.7+/-5.7 days, CI=0.084), MMF 10 mg/kg/day+RAPA 0.4 mg(kg/day (50.2+/-13.5 days, CI=0.453), and MMF 20 mg/kg(day+ RAPA 0.4 mg/kg(day (51.5+/-6.8 days, CI=0.439), respectively. These results were repeatable in the prevention of acute pancreas and kidney allograft rejection in the rat. In the second part of the study of reversal of ongoing acute heart allograft rejection model, the combined treatment of MMF 10 mg/kg(day+RAPA 0.2 mg/ kg(day (35.5+/-16.0 days, CI=0.794) and MMF 20 mg/kg day+RAPA 0.2 mg(kg/day (57.2+/-4.7 days, CI=0.310) represented synergistic interaction compared with monotherapy of MMF or RAPA. CONCLUSIONS Concomitant therapy of MMF and RAPA produces a synergistic effect in prevention of heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat.

Compromised kidney graft rejection response in Vervet monkeys after withdrawal of immunosuppressants tacrolimus and sirolimus.

BACKGROUND In nonprimates, organ allografts are often not rejected after withdrawal of immunosuppression. In this study, we examined whether such a phenomenon also occurs in primates. METHODS Vervet monkeys were transplanted with renal allografts and treated for 60 days with tacrolimus, or tacrolimus plus sirolimus. The drugs were totally withdrawn on day 61. The survival of the monkeys was monitored, and their response to donor- or third party-derived alloantigens was examined in vivo and in vitro. RESULTS The majority (80-100%) of the grafts survived for at least additional 30 days with no signs of acute rejection. The compromised rejection is donor-specific, because recipient monkeys failed to reject a donor-derived skin graft, but a third-party skin graft was rejected. In vitro mixed lymphocyte reaction and interleukin-2 production in the mixed lymphocyte reaction between the recipients and their donors or between the recipients and a third party had no discernable patterns, and thus did not reflect the in vivo status of the immune system. Although the recipients could not reject the graft acutely after drug withdrawal, the kidney grafts and the donor-derived skin grafts had pathological findings of chronic rejection. CONCLUSIONS The rejection response of the monkeys to an established graft after withdrawal of immunosuppression is compromised. The compromised rejection is specific and is not due to a permanent alteration of the immune system by the initial drug treatment. The allografts are not inert but have low levels of interaction with the recipient immune system.

Receptor tyrosine kinase Ephb6 regulates vascular smooth muscle contractility and modulates blood pressure in concert with sex hormones.

Background: Eph kinases constitute the largest receptor tyrosine kinase family, and there is no knowledge about their function in blood pressure regulation. Results: Ephb6 regulates vascular smooth muscle cell contraction, and its knock-out resulted in increased blood pressure in castrated male mice. Conclusion: Ephb6 and its ligands can regulate vessel tone and blood pressure. Significance: We have identified a new group of molecules capable of regulating blood pressure. Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.

Possible Role of Efnb1 Protein, a Ligand of Eph Receptor Tyrosine Kinases, in Modulating Blood Pressure

Background: Currently, there is no knowledge about the function of ephrins in vascular smooth muscle contraction and blood pressure regulation. Results: Stimulating Efnb1 reduces vascular smooth muscle cell contraction. Efnb1 null mutation increases RhoA activation and heightens blood pressure in mice. Conclusion: Efnb1 can regulate vessel tone and blood pressure. Significance: We identified a new group of molecules capable of regulating blood pressure. Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Although they are ligands, Efns can transduce signals reversely into cells. We have no prior knowledge of the role played by any members of this family of kinases or their ligands in blood pressure (BP) regulation. In the present studies, we investigated the role of Efnb1 in vascular smooth muscle cell (VSMC) contractility and BP regulation. We revealed that reverse signaling through Efnb1 led to a reduction of RhoA activation and VSMC contractility in vitro. Consistent with this finding, ex vivo, there was an increase of RhoA activity accompanied by augmented myosin light chain phosphorylation in mesenteric arteries from mice with smooth muscle-specific conditional Efnb1 gene knock-out (KO). Small interfering RNA knockdown of Grip1, a molecule associated with the Efnb1 intracellular tail, partially eliminated the effect of Efnb1 on VSMC contractility and myosin light chain phosphorylation. In support of these in vitro and ex vivo results, Efnb1 KO mice on a high salt diet showed a statistically significant heightened increment of BP at multiple time points during stress compared with wild type littermates. Our results demonstrate that Efnb1 is a previously unknown negative regulator of VSMC contractility and BP and that it exerts such effects via reverse signaling through Grip1.

Blocking of CD44-hyaluronic acid interaction prolongs rat allograft survival.

BACKGROUND Lymphocyte activation and infiltration into a transplanted organ is an integral component of the rejection process. Graft infiltration of lymphocytes requires adhesion of leukocytes to the endothelium, diapedesis, and transmigration. One of several proteins involved in this process is CD44, which is known to interact with endothelial hyaluronan (HA). Blockade of cell-matrix and cell-cell interactions have been used extensively for modulation of immune responses and graft rejection. Based on these observations, we evaluated the effects of blocking CD44-HA interactions in a transplantation model. METHODS We used a low molecular weight hyaluronic acid formulation (LMWHA) for the treatment of rat renal and cardiac allograft recipients. LMWHA was administered intraperitoneally at 0.5-5 mg/kg for 5-10 days after transplantation with or without a subtherapeutic dose of cyclosporine. RESULTS LMWHA monotherapy prolonged allograft survival significantly, but only for a few days. In combination with low-dose cyclosporine, long-term survival of allografts was observed in some of recipients. CONCLUSION Further definition of the underlying mechanism of LMWHA therapy may provide a rationale for the development of novel, nontoxic, nonimmunogenic immunotherapies.

EPHB4 Protein Expression in Vascular Smooth Muscle Cells Regulates Their Contractility, and EPHB4 Deletion Leads to Hypotension in Mice

Background: The role of EPHB4 blood pressure regulation was not previously known. Results: Male but not female smooth muscle cell-specific Ephb4 gene knock-out mice were hypotensive; bi-directional signaling between EFNBs and EPHB4 modulated small artery contractility. Conclusion: EPHB4 in vascular smooth muscle cells regulates blood pressure. Significance: A new mechanism of blood pressure regulation has been discovered. EPH kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. This work presents evidence that EPHB4 on vascular smooth muscle cells (VSMCs) is involved in blood pressure regulation. We generated gene KO mice with smooth muscle cell-specific deletion of EPHB4. Male KO mice, but not female KO mice, were hypotensive. VSMCs from male KO mice showed reduced contractility when compared with their WT counterparts. Signaling both from EFNBs to EPHB4 (forward signaling) and from EPHB4 to EFNB2 (reverse signaling) modulated VSMC contractility. At the molecular level, the absence of EPHB4 in VSMCs resulted in compromised signaling from Ca2+/calmodulin-dependent protein kinase II (CaMKII) to myosin light chain kinase (MLCK) to myosin light chain, the last of which controls the contraction force of motor molecule myosin. Near the cell membrane, an adaptor protein GRIP1, which can associate with EFNB2, was found to be essential in mediating EPHB4-to-EFNB reverse signaling, which regulated VSMC contractility, based on siRNA gene knockdown studies. Our research indicates that EPHB4 plays an essential role in regulating small artery contractility and blood pressure.

Serine carboxypeptidase SCPEP1 and Cathepsin A play complementary roles in regulation of vasoconstriction via inactivation of endothelin-1.

The potent vasoconstrictor peptides, endothelin 1 (ET-1) and angiotensin II control adaptation of blood vessels to fluctuations of blood pressure. Previously we have shown that the circulating level of ET-1 is regulated through its proteolytic cleavage by secreted serine carboxypeptidase, cathepsin A (CathA). However, genetically-modified mouse expressing catalytically inactive CathA S190A mutant retained about 10–15% of the carboxypeptidase activity against ET-1 in its tissues suggesting a presence of parallel/redundant catabolic pathway(s). In the current work we provide direct evidence that the enzyme, which complements CathA action towards ET-1 is a retinoid-inducible lysosomal serine carboxypeptidase 1 (Scpep1), a CathA homolog with previously unknown biological function. We generated a mouse strain devoid of both CathA and Scpep1 activities (DD mice) and found that in response to high-salt diet and systemic injections of ET-1 these animals showed significantly increased blood pressure as compared to wild type mice or those with single deficiencies of CathA or Scpep1. We also found that the reactivity of mesenteric arteries from DD mice towards ET-1 was significantly higher than that for all other groups of mice. The DD mice had a reduced degradation rate of ET-1 in the blood whereas their cultured arterial vascular smooth muscle cells showed increased ET-1-dependent phosphorylation of myosin light chain 2. Together, our results define the biological role of mammalian serine carboxypeptidase Scpep1 and suggest that Scpep1 and CathA together participate in the control of ET-1 regulation of vascular tone and hemodynamics.

Estrogen and testosterone in concert with EFNB3 regulate vascular smooth muscle cell contractility and blood pressure.

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions, although their function in blood pressure (BP) control has not been studied in detail. In the present study, we report that Efnb3 gene knockout (KO) led to increased BP in female but not male mice. Vascular smooth muscle cells (VSMCs) were target cells for EFNB3 function in BP regulation. The deletion of EFNB3 augmented contractility of VSMCs from female but not male KO mice, compared with their wild-type (WT) counterparts. Estrogen augmented VSMC contractility while testosterone reduced it in the absence of EFNB3, although these sex hormones had no effect on the contractility of VSMCs from WT mice. The effect of estrogen on KO VSMC contractility was via a nongenomic pathway involving GPER, while that of testosterone was likely via a genomic pathway, according to VSMC contractility assays and GPER knockdown assays. The sex hormone-dependent contraction phenotypes in KO VSMCs were reflected in BP in vivo. Ovariectomy rendered female KO mice normotensive. At the molecular level, EFNB3 KO in VSMCs resulted in reduced myosin light chain kinase phosphorylation, an event enhancing sensitivity to Ca(2+)flux in VSMCs. Our investigation has revealed previously unknown EFNB3 functions in BP regulation and show that EFNB3 might be a hypertension risk gene in certain individuals.