Increased Prolylcarboxypeptidase Expression Can Serve as a Biomarker of Senescence in Culture.

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

Potential Benefits of Antioxidant Phytochemicals in Type 2 Diabetes.

The clinical relationship between diabetes and inflammation is well established. Evidence clearly indicates that disrupting oxidant-antioxidant equilibrium and elevated lipid peroxidation could be a potential mechanism for chronic kidney disease associated with type 2 diabetes mellitus (T2DM). Under diabetic conditions, hyperglycemia, especially inflammation, and increased reactive oxygen species generation are bidirectionally associated. Inflammation, oxidative stress, and tissue damage are believed to play a role in the development of diabetes. Although the exact mechanism underlying oxidative stress and its impact on diabetes progression remains uncertain, the hyperglycemia-inflammation-oxidative stress interaction clearly plays a significant role in the onset and progression of vascular disease, kidney disease, hepatic injury, and pancreas damage and, therefore, holds promise as a therapeutic target. Evidence strongly indicates that the use of multiple antidiabetic medications fails to achieve the normal range for glycated hemoglobin targets, signifying treatment-resistant diabetes. Antioxidants with polyphenols are considered useful as adjuvant therapy for their potential anti-inflammatory effect and antioxidant activity. We aimed to analyze the current major points reported in preclinical, in vivo, and clinical studies of antioxidants in the prevention or treatment of inflammation in T2DM. Then, we will share our speculative vision for future diabetes clinical trials.

Identification of Human Kinin-Forming Enzyme Inhibitors from Medicinal Herbs.

The goal of this study was to assess the pharmacological effects of black tea (Camellia sinensis var. assamica) water extract on human kinin-forming enzymes in vitro. Tea is a highly consumed beverage in the world. Factor XII (FXII, Hageman factor)-independent- and -dependent activation of prekallikrein to kallikrein leads to the liberation of bradykinin (BK) from high-molecular-weight kininogen (HK). The excessive BK production causes vascular endothelial and nonvascular smooth muscle cell permeability, leading to angioedema. The prevalence of angiotensin-converting enzyme inhibitor (ACEI)-induced angioedema appears to be through BK. Both histamine and BK are potent inflammatory mediators. However, the treatments for histamine-mediated angioedema are unsuitable for BK-mediated angioedema. We hypothesized that long-term consumption of tea would reduce bradykinin-dependent processes within the systemic and pulmonary vasculature, independent of the anti-inflammatory actions of polyphenols. A purified fraction of the black tea water extract inhibited both kallikrein and activated FXII. The black tea water extracts inhibited factor XII-induced cell migration and inhibited the production of kallikrein on the endothelial cell line. We compared the inhibitory effects of the black tea water extract and twenty-three well-known anti-inflammatory medicinal herbs, in inhibiting both kallikrein and FXII. Surprisingly, arjunglucoside II specifically inhibited the activated factor XII (FXIIa), but not the kallikrein and the activated factor XI. Taken together, the black tea water extract exerts its anti-inflammatory effects, in part, by inhibiting kallikrein and activated FXII, which are part of the plasma kallikrein-kinin system (KKS), and by decreasing BK production. The inhibition of kallikrein and activated FXII represents a unique polyphenol-independent anti-inflammatory mechanism of action for the black tea.

Pregnane steroidogenesis is altered by HIV-1 Tat and morphine: Physiological allopregnanolone is protective against neurotoxic and psychomotor effects.

Pregnane steroids, particularly allopregnanolone (AlloP), are neuroprotective in response to central insult. While unexplored in vivo, AlloP may confer protection against the neurological dysfunction associated with human immunodeficiency virus type 1 (HIV-1). The HIV-1 regulatory protein, trans-activator of transcription (Tat), is neurotoxic and its expression in mice increases anxiety-like behavior; an effect that can be ameliorated by progesterone, but not when 5α-reduction is blocked. Given that Tat's neurotoxic effects involve mitochondrial dysfunction and can be worsened with opioid exposure, we hypothesized that Tat and/or combined morphine would perturb steroidogenesis in mice, promoting neuronal death, and that exogenous AlloP would rescue these effects. Like other models of neural injury, conditionally inducing HIV-1 Tat in transgenic mice significantly increased the central synthesis of pregnenolone and progesterone's 5α-reduced metabolites, including AlloP, while decreasing central deoxycorticosterone (independent of changes in plasma). Morphine significantly increased brain and plasma concentrations of several steroids (including progesterone, deoxycorticosterone, corticosterone, and their metabolites) likely via activation of the hypothalamic-pituitary-adrenal stress axis. Tat, but not morphine, caused glucocorticoid resistance in primary splenocytes. In neurons, Tat depolarized mitochondrial membrane potential and increased cell death. Physiological concentrations of AlloP (0.1, 1, or 10 nM) reversed these effects. High-concentration AlloP (100 nM) was neurotoxic in combination with morphine. Tat induction in transgenic mice potentiated the psychomotor effects of acute morphine, while exogenous AlloP (1.0 mg/kg, but not 0.5 mg/kg) was ameliorative. Data demonstrate that steroidogenesis is altered by HIV-1 Tat or morphine and that physiological AlloP attenuates resulting neurotoxic and psychomotor effects.

Dynorphins in Development and Disease: Implications for Cardiovascular Disease.

It is well-established that cardiovascular disease continues to represent a growing health problem and significant effort has been made to elucidate the underlying mechanisms. In this review, we report on past and recent high impact publications in the field of intracrine network signaling, focusing specifically on opioids and their interrelation with key modulators of the cardiovascular system and the onset of related disease. We present an overview of studies outlining the scope of cardiovascular and cerebrovascular processes that are affected by opioids, including heart function, ischemia, reperfusion, and blood flow. Specific emphasis is placed on the importance of dynorphin molecules in cerebrovascular and cardiovascular regulation. Evidence suggests that excessive or insufficient dynorphin could make an important contribution to cardiovascular physiology, yet numerous paradoxical observations frequently impede a clear understanding of the role of dynorphin. Thus, we argue that dynorphin-mediated signaling events for which an immediate regulatory effect is disputed should not be dismissed as unimportant, as they may play a role in cross-talk with other signaling networks. Finally, we consider the most recent evidence on the role of dynorphin during cardiovascular-related inflammation and on the potential value of endogenous and exogenous inhibitors of kappa-opioid receptor, a major dynorphin A receptor, to limit or prevent cardiovascular disease and its related sequelae.

4-O-Methylhonokiol Influences Normal Cardiovascular Development in Medaka Embryo.

Although 4-O-Methylhonokiol (MH) effects on neuronal and immune cells have been established, it is still unclear whether MH can cause a change in the structure and function of the cardiovascular system. The overarching goal of this study was to evaluate the effects of MH, isolated from Magnolia grandiflora, on the development of the heart and vasculature in a Japanese medaka model in vivo to predict human health risks. We analyzed the toxicity of MH in different life-stages of medaka embryos. MH uptake into medaka embryos was quantified. The LC50 of two different exposure windows (stages 9⁻36 (0⁻6 days post fertilization (dpf)) and 25⁻36 (2⁻6 dpf)) were 5.3 ± 0.1 µM and 9.9 ± 0.2 µM. Survival, deformities, days to hatch, and larval locomotor response were quantified. Wnt 1 was overexpressed in MH-treated embryos indicating deregulation of the Wnt signaling pathway, which was associated with spinal and cardiac ventricle deformities. Overexpression of major proinflammatory mediators and biomarkers of the heart were detected. Our results indicated that the differential sensitivity of MH in the embryos was developmental stage-specific. Furthermore, this study demonstrated that certain molecules can serve as promising markers at the transcriptional and phenotypical levels, responding to absorption of MH in the developing embryo.

Free Fatty Acids: Circulating Contributors of Metabolic Syndrome.

Metabolic syndrome induces an increased cardiovascular morbidity and mortality. Most importantly, the prevalence of metabolic syndrome in adult population is expanding. Both clinical and preclinical studies indicate that increased Free Fatty Acids (FFAs) are involved in the pathogenesis of insulin resistance and subsequent development of metabolic syndrome. The relevance of FFAs in protecting and restoring tissue function is quite vast. The search to correlate the functional deterioration of the tissues within the cardiovascular system and increased plasma concentrations of FFAs has been reported. The importance of reduction in the consumption of dietary fatty acids along with the identification of dysregulated genes responsible for persistent increased FFAs uptake and mitochondrial ß-oxidation has been increasingly recognized. This review discusses the current empirical understanding of the different types of fatty acids and their metabolism and functions both in physiological and pathophysiological conditions. We also discuss in detail about the molecular and pathophysiological basis of increased FFAs, which augments Cardiovascular Disease (CVD).

Altered Prolylcarboxypeptidase Expression and Function in Response to Different Risk Factors of Diabetes.

BACKGROUND: Prolylcarboxypeptidase (PRCP, EC:3.4.16.2) is a cardioprotective protease. Plasma PRCP levels are elevated in type 2 diabetes (T2D) mellitus and cardiovascular diseases. OBJECTIVE: Since diabetic cardiomyopathy is a late complication of uncontrolled diabetes, we tested the hypothesis that glucose and free fatty acid related risk factors for T2D mellitus and cardiovascular disease may reduce the cardioprotective property of PRCP. METHOD: We examined the effects of glucose, saturated fatty acids, and unsaturated fatty acids on PRCP expression in cultured H9c2 cells as an in-vitro model for pharmacological studies. Selective inhibitors, known cardioprotective agents and saturating amounts of neutralizing antibodies were used to validate the effect of free fatty acids on the expression and function of PRCP. RESULTS: The palmitate-mediated reduction of PRCP was concentration and time-dependent. Next, we explored the cardioprotective potential of thyroxin (T4) and insulin. Both T4 and insulin were able to prevent the palmitate-mediated reduction of PRCP expression in H9c2 cells. Inhibition of NF-kB with its specific inhibitor Bay 11-7082 or blockade of palmitate with polyunsaturated fatty acids was ineffective in preventing palmitate-mediated decreases in PRCP expression. CONCLUSION: Our data indicate that elevated palmitate inhibits PRCP expression in rat cardiomyocyte. From this inference PRCP level should be monitored in obese or diabetic patients because this simple measure could identify individuals at high risk of developing health problems, such as heart failure.

Mechanism for neurotropic action of vorinostat, a pan histone deacetylase inhibitor.

In this study we investigated the neurotrophic actions of vorinostat (suberoylanilide hydroxamic acid, SAHA), a class I and class II HDAC inhibitor, on the differentiation of Neuroscreen-1 (NS-1) cells. NS-1 cell is a subclone of the rat pheochromocytoma cell line (PC 12). Vorinostat independently induced neurite outgrowth in NS-1 cells. The NS-1 cells were further interrogated for the effects of vorinostat on intracellular neurotrophin signaling pathways, to understand its mechanism of neurotrophic action. Selective inhibitors of MEK1/2 (PD98059 and U0126), phosphoinositide 3-kinase (PI3K) (LY294002) and tyrosine kinase A (TrkA) (GW441756) were employed for these interrogations. Our results suggest that neurite outgrowth mediated by both nerve growth factor (NGF), an intrinsic neurotrophin, and vorinostat were blocked by the inhibitors of MEK1/2 & PI3K. Vorinostat induced phosphorylation of ERK1/2 occurs at 2h post treatment. Phosphorylation of ERK was abolished in presence of U0126, further confirming the role of ERK pathway in vorinostat-induced differentiation of NS-1 cells. Vorinostat-induced neurite outgrowth also involves the activation of upstream extracellular kinase TrkA, as both vorinostat mediated neurite outgrowth and activation of ERK were attenuated in presence of the TrkA inhibitor, GW441756. Vorinostat also stimulated hyperacetylation of α-tubulin and histones H3/H4 in NS-1 cells. The results suggest that vorinostat exerts a positive effect on the neuritogenesis via activation of MEK1/2 & PI3K pathways involving an upstream kinase, TrkA. Bioactive small molecules with neurotrophic and neuritogenic actions, like vorinostat identified in the present study, hold great promise as therapeutic agents for treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth.

The anticancer potential of steroidal saponin, dioscin, isolated from wild yam (Dioscorea villosa) root extract in invasive human breast cancer cell line MDA-MB-231 in vitro.

Previously, we observed that wild yam (Dioscorea villosa) root extract (WYRE) was able to activate GATA3 in human breast cancer cells targeting epigenome. This study aimed to find out if dioscin (DS), a bioactive compound of WYRE, can modulate GATA3 functions and cellular invasion in human breast cancer cells. MCF-7 and MDA-MB-231 cells were treated in the absence/presence of various concentrations of DS and subjected to gene analysis by RT-qPCR, immunoblotting, and immunocytochemistry. We determined the ability of MDA-MB-231 cells to migrate into wound area and examined the effects of DS on cellular invasion using invasion assay. DS reduced cell viability of both cell lines in a concentration and time-dependent manner. GATA3 expression was enhanced by DS (5.76 µM) in MDA-MB-231 cells. DS (5.76 µM)-treated MDA-MB-231 cells exhibited the morphological characteristic of epithelial-like cells; mRNA expression of DNMT3A, TET2, TET3, ZFPM2 and E-cad were increased while TET1, VIM and MMP9 were decreased. Cellular invasion of MDA-MB-231 was reduced by 65 ± 5% in the presence of 5.76 µM DS. Our data suggested that DS-mediated pathway could promote GATA3 expression at transcription and translation levels. We propose that DS has potential to be used as an anti-invasive agent in breast cancer.

Plasma Kallikrein Inhibitors in Cardiovascular Disease: An Innovative Therapeutic Approach.

Plasma prekallikrein is the liver-derived precursor of the trypsin-like serine protease plasma kallikrein, and circulates in plasma bound to high molecular weight kininogen. Plasma prekallikrein is activated to plasma kallikrein by activated factor XII or prolylcarboxypeptidase. Plasma kallikrein regulates the activity of multiple proteolytic cascades in the cardiovascular system such as the intrinsic pathway of coagulation, the kallikrein-kinin system, the fibrinolytic system, the renin-angiotensin system, and the complement pathways. As such, plasma kallikrein plays a central role in the pathogenesis of thrombosis, inflammation, and blood pressure regulation. Under physiological conditions, plasma kallikrein serves as a cardioprotective enzyme. However, its increased plasma concentration or hyperactivity perpetuates cardiovascular disease (CVD). In this article, we review the biochemistry and cell biology of plasma kallikrein and summarize data from preclinical and clinical studies that have established important functions of this serine protease in CVD states. Finally, we propose plasma kallikrein inhibitors as a novel class of drugs with potential therapeutic applications in the treatment of CVDs.

Prolylcarboxypeptidase gene expression in the heart and kidney: Effects of obesity and diabetes.

Prolylcarboxypeptidase (PRCP) regulates plasma prekallikrein/high molecular weight kininogen/bradykinin axis. It also modulates angiotensin II (Ang II), angiotensin III (Ang III), and alpha-melanocyte stimulating hormone (α-MSH) physiological effects. Study suggests that increased plasma PRCP level is associated with cardiovascular risk factors, such as atherosclerosis, inflammation, and diabetes. Since expression pattern of PRCP in Zucker diabetic fatty (ZDF) rat vascular tissue remain unproved, we aimed to study its expression in the heart and kidney. The purpose of the present study was also to obtain systemic information of inflammation status with regard to PRCP expression and function in a high-fat diet (HFD)- fed ZDF rats. The ZDF rats were divided into 2 groups, which were fed a high-fat diet for 16 weeks or 32 weeks. Differential expression and pathological significance of PRCP expression during the consecutive stages of renal disease development were identified. After 16 weeks, ZDF rats exhibited early transiently altered PRCP expression in the heart and kidneys. After 32 weeks, ZDF rats showed continuously altered expression in PRCP and inflammatory markers, which was linked to severe hyperglycemia and nephropathy. Altered expression of PRCP associated with inflammatory mediators was illustrated to be functionally relevant. In further support of an important role of PRCP, we found PRCP protein to be highly elevated in rat plasma and in human plasma and the anti-diabetic agents reversed it. These findings indicate that impairment of tissues within the cardiovascular system influences PRCP expression and suggest that pathogenic mechanisms of deregulated PRCP expression warrant further investigation.

Novel Redox-Responsive Amphiphilic Copolymer Micelles for Drug Delivery: Synthesis and Characterization.

A novel redox-responsive amphiphilic polymer was synthesized with bioreductive trimethyl-locked quinone propionic acid for a potential triggered drug delivery application. The aim of this study was to synthesize and characterize the redox-responsive amphiphilic block copolymer micelles containing pendant bioreductive quinone propionic acid (QPA) switches. The redox-responsive hydrophobic block (polyQPA), synthesized from QPA-serinol and adipoyl chloride, was end-capped with methoxy poly(ethylene glycol) of molecular weight 750 (mPEG750) to achieve a redox-responsive amphiphilic block copolymer, polyQPA-mPEG750. PolyQPA-mPEG750 was able to self-assemble as micelles to show a critical micelle concentration (CMC) of 0.039% w/v (0.39 mg/ml, 0.107 mM) determined by a dye solubilization method using 1,6-diphenyl-1,3,5-hexatriene (DPH) in phosphate-buffered saline (PBS). The mean diameter of polymeric micelles was found to be 27.50 nm (PI = 0.064) by dynamic light scattering. Furthermore, redox-triggered destabilization of the polymeric micelles was confirmed by (1)H-NMR spectroscopy and particle size measurements in a simulated redox state. PolyQPA-mPEG750 underwent triggered reduction to shed pendant redox-responsive QPA groups and its polymeric micelles were swollen to be dissembled in the presence of a reducing agent, thereby enabling the release of loaded model drug, paclitaxel. The redox-responsive polyQPA-mPEG750 polymer micelles would be useful as a drug delivery system allowing triggered drug release in an altered redox state such as tumor microenvironments with an altered redox potential and/or redox enzyme upregulation.

The prolyl peptidases PRCP/PREP regulate IRS-1 stability critical for rapamycin-induced feedback activation of PI3K and AKT.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells.

Domain 2 of uPAR regulates single-chain urokinase-mediated angiogenesis through ß1-integrin and VEGFR2.

How single-chain urokinase (ScuPA) mediates angiogenesis is incompletely understood. ScuPA (≥4 nM) induces phosphorylated (p)ERK1/2 (MAPK44 and MAPK42) and pAkt (Ser(473)) in umbilical vein and dermal microvascular endothelial cells. Activation of pERK1/2 by ScuPA is blocked by PD-98059 or U-0126, and pAkt (Ser(473)) activation is inhibited by wortmannin or LY-294002. ScuPA (32 nM) or protease-inhibited two-chain urokinase stimulates pERK1/2 to the same extent, indicating that signaling is not dependent on enzymatic activity. ScuPA induces pERK1/2, but not pAkt (Ser(473)), in SIN1(-/-) cells, indicating that the two pathways are not identical. Peptides from domain 2 of the urokinase plasminogen activator receptor (uPAR) or domain 5 of high-molecular-weight kininogen compete with ScuPA for the induction of pERK1/2 and pAkt (Ser(473)). A peptide of the integrin-binding site on uPAR, a ß1-integrin peptide that binds uPAR, antibody 6S6 to ß1-integrin, tyrosine kinase inhibitors AG-1478 or PP3, and small interfering RNA knockdown of VEFG receptor 2, but not HER1-HER4, blocked ScuPA-induced pERK1/2 and pAkt (Ser(473)). ScuPA-induced endothelial cell proliferation was blocked by inhibitors of pERK1/2 and pAkt (Ser(473)), antibody 6S6, and uPAR or kininogen peptides. ScuPA initiated aortic sprouts and Matrigel plug angiogenesis in normal, but not uPAR-deficient, mouse aortae or mice, respectively, but these were blocked by PD-98059, LY-294002, AG-1478, or cleaved high-molecular-weight kininogen. In summary, this investigation indicates a novel, a nonproteolytic signaling pathway initiated by zymogen ScuPA and mediated by domain 2 of uPAR, ß1-integrins, and VEGF receptor 2 leading to angiogenesis. Kininogens or peptides from it downregulate this pathway.

High molecular weight kininogen activates B2 receptor signaling pathway in human vascular endothelial cells.

The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca(2+)](i) level in HPAEC. Gd(3+) and verapamil potentiated the HK-induced increase in [Ca(2+)](i). HK-induced Ca(2+) increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B(2) receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.

Murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis.

Prolylcarboxypeptidase (PRCP) activates prekallikrein to plasma kallikrein, leading to bradykinin liberation, and degrades angiotensin II. We now identify PRCP as a regulator of blood vessel homeostasis. ß-Galactosidase staining in PRCP(gt/gt) mice reveals expression in kidney and vasculature. Invasive telemetric monitorings show that PRCP(gt/gt) mice have significantly elevated blood pressure. PRCP(gt/gt) mice demonstrate shorter carotid artery occlusion times in 2 models, and their plasmas have increased thrombin generation times. Pharmacologic inhibition of PRCP with Z-Pro-Prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-chloromethylketone or PKSI 527 also shortens carotid artery occlusion times. Aortic and renal tissues have uncoupled eNOS and increased reactive oxygen species (ROS) in PRCP(gt/gt) mice as detected by dihydroethidium or Amplex Red fluorescence or lucigenin luminescence. The importance of ROS is evidenced by the fact that treatment of PRCP(gt/gt) mice with antioxidants (mitoTEMPO, apocynin, Tempol) abrogates the hypertensive, prothrombotic phenotype. Mechanistically, our studies reveal that PRCP(gt/gt) aortas express reduced levels of Kruppel-like factors 2 and 4, thrombomodulin, and eNOS mRNA, suggesting endothelial cell dysfunction. Further, PRCP siRNA treatment of endothelial cells shows increased ROS and uncoupled eNOS and decreased protein C activation because of thrombomodulin inactivation. Collectively, our studies identify PRCP as a novel regulator of vascular ROS and homeostasis.

Prolylcarboxypeptidase regulates proliferation, autophagy, and resistance to 4-hydroxytamoxifen-induced cytotoxicity in estrogen receptor-positive breast cancer cells.

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.

Factor XII stimulates ERK1/2 and Akt through uPAR, integrins, and the EGFR to initiate angiogenesis.

Factor XII (FXII) and high molecular weight kininogen (HK) mutually block each other's binding to the urokinase plasminogen activator receptor (uPAR). We investigated if FXII stimulates cells by interacting with uPAR. FXII (3-62nM) with 0.05mM Zn(2+) induces extracellular signal-related kinase 1/2 (ERK1/2; mitogen-activated protein kinase 44 [MAPK44] and MAPK42) and Akt (Ser473) phosphorylation in endothelial cells. FXII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. An uPAR signaling region for FXII is on domain 2 adjacent to uPAR's integrin binding site. Cleaved HK or peptides from HK's domain 5 blocks FXII-induced ERK1/2 and Akt phosphorylation. A beta(1) integrin peptide that binds uPAR, antibody 6S6 to beta(1) integrin, or the epidermal growth factor receptor (EGFR) inhibitor AG1478 blocks FXII-induced phosphorylation of ERK1/2 and Akt. FXII induces endothelial cell proliferation and 5-bromo-2'deoxy-uridine incorporation. FXII stimulates aortic sprouting in normal but not uPAR-deficient mouse aorta. FXII produces angiogenesis in matrigel plugs in normal but not uPAR-deficient mice. FXII knockout mice have reduced constitutive and wound-induced blood vessel number. In sum, FXII initiates signaling mediated by uPAR, beta(1) integrin, and the EGFR to induce human umbilical vein endothelial cell proliferation, growth, and angiogenesis.