Human Tissue Kallikrein Research Articles

Multianalyte quantitative competitive PCR on optically encoded microspheres for an eight-gene panel related to prostate cancer.

Nucleic acid-based tests have a profound impact in every medical discipline. Because multigene tests offer higher diagnostic accuracy and lower overall cost than single assays, they are especially useful for diseases, like prostate cancer, that present variability at the molecular level and diversity of available therapeutic interventions. We have developed a quantitative competitive PCR for an eight-gene panel, related to prostate cancer, that includes five genes of the human tissue kallikrein family (KLKs), prostate-specific membrane antigen (PSMA), prostate cancer antigen 3 (PCA3), and HPRT1 as a reference gene. Using PCR as a synthetic tool, a competitor was prepared for each target sequence containing the same primer binding sites as the target but differing in a short segment to enable discrimination by hybridization. The assay involves multiplex amplification of targets and competitors followed by a multiplex hybridization assay for the 16 amplification products. The assay was performed on optically encoded microspheres with oligonucleotide probes attached to their surface. The microspheres were analyzed rapidly (1min) by flow cytometry. The signal ratio of the target and cognate competitor is a function of the target copy number in the sample prior to amplification. The multiplexing potential of the proposed method is much higher than real-time PCR and other end-point methods since there are 100 sets of commercially available microspheres.

AB087. Preserved erectile function in the hyperhomocysteinaemia transgenic rats harboring human tissue kallikrein

BackgroundTo investigate the role of human tissue kallikrein 1 (hKLK1) gene on the erectile dysfunction (ED) of induced by hyperhomocysteinaemia (HHcy) in rats.MethodsThe HHcy rat model was formed by a methionine (Met)-rich diet in SD rats. Here, 32 rats, 10-week-age, were divided in four groups: control (n=8), low-dose (4% Met; n=8), high-dose (7% Met; n=8) and transgenic rats (TGR +7% Met; n=8). Thirty days later, erectile function and related targets were tested.ResultsED, impaired endothelial and smooth muscle function, and pathological changes (a higher apoptosis level and a lower autophagy level) were showed in the 4% Met and 7% Met groups compared with the control, while were all markedly diminished by the hKLK1 gene in the TGR +7% Met group.ConclusionsThese data suggested that hKLK1 might play an inhibition role on HHcy-induced ED in rats by protection of endothelial function and inhibition of oxidative stress and corporal fibrosis.

Biochemical and functional characterization of the human tissue kallikrein 9.

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+ KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

Abstract 854: Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer

Abstract Background: Kallikrein-related peptidase 6 (KLK6) belongs to the family of human tissue kallikrein genes, majority of which are shown to be differentially expressed in cancers. Clinical studies have demonstrated that upregulation of KLK6 in primary colorectal tumors and lymph nodes correlates with serosal invasion, liver metastasis and indicative of poor prognosis in patients. It has been reported that KLK6 protein is involved in regulation of the epithelial-mesenchymal transition (EMT) program in an organ-specific context. The aim in this study was to investigate contribution of KLK6 enzyme in the EMT during neoplastic transformation in the colon. Results: We expressed enzymatically active or inactive KLK6, using pcDNA3.1(+)preproKLK6 and pcDNA3.1(+)preproKLK6 Ser197Ala mutant plasmids, in Caco-2 colon cancer cell line, which has been characterized before as a very low KLK6 expresser with an undetectable secreted KLK6. Stable isogenic clones were selected and further evaluated for their ability to migrate and invade using in vitro assays and to metastasize in vivo using SCID orthotopic mouse model. We found no effect of KLK6 enzyme activity on migration of Caco-2 cells, expressing the empty vector (Caco-2 mock), and Caco-2 cells, expressing an enzymatically active KLK6 (Caco-2 KLK6 wt) or inactive KLK6 (Caco-2 KLK6 mut). But Caco-2 KLK6 wt cells demonstrated the invasive phenotype in Matrigel invasion assays (p<0.001, compared to Caco-2 mock and Caco2 KLK6 mut cells). The Caco-2 mock and Caco-2 KLK6 mut cells, injected in SCID mice orthotopically, developed primary colon tumors but no metastatic lesions were identified. In contrast, Caco-2 KLK6 wt cells formed primary colon tumors and metastasized locally, although they failed to form the distant metastasis (lung and mesentery). Animals, growing the Caco-2 KLK6 wt tumors, displayed a significant decrease in their survival rates, compared to other groups (p=0.02). In Caco-2 KLK6 wt cells TGF-β protein expression and secretion was induced, which resulted in activation of TGF-β-SMAD2/3 signaling pathway. This phenotype was associated with the elevated expression of known regulator of the EMT, zinc-finger protein Snail. In addition, the expression of a high-mobility group AT-hook 2 (HMGA2) protein was induced in Caco-2 KLK6 wt cells. The HMGA2 expression is implicated in the EMT program, acting through the TGF-β signaling pathway and is associated with a poor survival in colorectal cancer. Conclusion. These findings demonstrate that KLK6 enzyme activity is required for colon cancer progression via induction of the EMT program. We identified the TGF-β- signaling pathway as a mechanism driving the EMT in colon cancer cells expressing KLK6 enzyme. Citation Format: Hwudaurw Chen, Earlphia Sells, Haiyan Cui, Ritu Pandey, George Pampalakis, Georgia Sotiropoulou, Thomas Doetschman, Natalia A. Ignatenko. Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 854. doi:10.1158/1538-7445.AM2017-854

Down-regulation of hK7 in the sera of breast cancer and benign breast disease patients.

IntroductionBreast cancer is known as a leading cause of cancer-related death among women all over the world. Biomarkers facilitate diagnosis at the earliest possible stage and better prognosis of the disease. Hence, may help to improve the overall survival rate among breast cancer patients. To find a better diagnostic/prognostic marker we evaluated human tissue kallikrein 7 (hK7) as biomarker of breast cancer. hK7 is a secreted serine protease having chymotrypsin like activity. Serum hK7 is known to have aberrant expression in ovarian and prostate cancer but has not been yet studied in breast cancer. However, the expression level of KLK7 mRNA in breast cancer tissues has been indicated as a better prognostic marker for the unfavorable prognosis of breast carcinoma.Materials and methodsIn this study a time-resolved immunofluorometric indirect back titration ELISA (bt-ELISA) was employed for the quantification of hK7 in serum of breast cancer patients (n = 47), benign breast disease patients (n = 13) alongwith the gender and age group specific controls (n = 99).ResultshK7 was significantly down-regulated in the sera of female breast cancer patients (p < 0.0001; Mean 0.704 ± 0.533 μg/L) and benign breast disease patients (p = 0.0008; Mean 0.651 ± 0.584) as compared to normal controls (Mean 1.665 ± 1.174 μg/L).ConclusionsDown regulation of hK7 suggests the possible role of this protein in natural course of breast cancer and benign breast diseases. Study should be extended on large-scale to confirm the potential of hK7 as biomarker of breast cancer.

MP81-14 HUMAN TISSUE KALLIKREIN 1 AMELIORATES ERECTILE FUNCTION VIA MODULATING AUTOPHAGY AND ACTIVATING HIF-1?/COX-2 PATHWAY IN AGED TRANSGENIC RATS

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology II1 Apr 2017MP81-14 HUMAN TISSUE KALLIKREIN 1 AMELIORATES ERECTILE FUNCTION VIA MODULATING AUTOPHAGY AND ACTIVATING HIF-1?/COX-2 PATHWAY IN AGED TRANSGENIC RATS Zhe Tang, Kai Cui, Yang Luan, Yajun Ruan, Tao Wang, Jun Yang, Shaogang Wang, and Jihong Liu Zhe TangZhe Tang More articles by this author , Kai CuiKai Cui More articles by this author , Yang LuanYang Luan More articles by this author , Yajun RuanYajun Ruan More articles by this author , Tao WangTao Wang More articles by this author , Jun YangJun Yang More articles by this author , Shaogang WangShaogang Wang More articles by this author , and Jihong LiuJihong Liu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2540AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our previous studies have demonstrated that Human Tissue Kallikrein 1 (hKLK1) improved erectile function via several signaling pathways related to such as oxidative stress or corporal cavernosal fibrosis. However, the potential molecular mechanisms of hKLK1 inhibited age-related erectile dysfunction via modulating autophagy remains unknown. The aim of this article is to partly clarify the mechanisms of hKLK1 improving the erectile function in aged rats. METHODS Male wild-type Sprague-Dawley rats (WTR) and transgenic rats expressing the hKLK1 gene (TGR) were fed to 4 and 18 months of age, respectively, and divided into three groups: young WTR (yWTR) as the control, aged WTR (aWTR) and aged TGR (aTGR). Cavernous nerve electrostimulation was used to evaluate the erectile function of all rats. Transmission electron microscopy, immunohistochemistry, and western blotting were performed to determine the levels of autophagy. Related signaling pathways were detected by western blot and immunohistochemistry. RESULTS Compared with the yWTR group and aTGR group, the aWTR group showed (1)lower erectile function: lower intracavernosal pressure(ICP)/mean arterial pressure(MAP) ratio; (2) reduced expressions of eNOS and nNOS, lower NO level; (3) inhibited autophagy: decreased autophagosomes, lower expressions of BECN1 and LC3-II; and (4) low expression levels of PI3K/Akt/mTOR and Hif-1a/Cox-2 pathways. CONCLUSIONS The hKLK1 gene played a potential role of restoring erectile function in aged transgenic rats through modulating autophagy via PI3K/Akt/mTOR and Hif-1a/Cox-2 pathways. This finding provided evidence for hKLK1 being gene therapy method of age-related erectile dysfunction. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1094 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Zhe Tang More articles by this author Kai Cui More articles by this author Yang Luan More articles by this author Yajun Ruan More articles by this author Tao Wang More articles by this author Jun Yang More articles by this author Shaogang Wang More articles by this author Jihong Liu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1121 - Preserved erectile function in the hyperhomocysteinaemia transgenic rat harboring human tissue kallikrein 1

1121 - Preserved erectile function in the hyperhomocysteinaemia transgenic rat harboring human tissue kallikrein 1

Involvement of DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP Pathways in Human Tissue Kallikrein 1 Protecting Erectile Function in Aged Rats.

Our previous studies had reported that Human Tissue Kallikrein 1 (hKLK1) preserved erectile function in aged transgenic rats, while the detailed mechanism of hKLK1 protecting erectile function in aged rats through activation of cGMP and cAMP was not mentioned. To explore the latent mechanism, male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 4 and 18 months old and divided into four groups: young WTR (yWTR) as the control, aged WTR (aWTR), aged TGR (aTGR) and aged TGRs with HOE140 (aTGRH). Erectile function of all rats was evaluated by cavernous nerve electrostimulation method and measured by the ratio of intracavernous pressure/ mean arterial pressure (ICP/MAP) in rats. Expression levels of cAMP and cGMP were assessed, and related signaling pathways were detected by western blot, immunohistochemistry and RT-PCR. Our experiment results showed erectile function of the aWTR group and aTGRH group was lower compared with those of other two groups. Also, expression levels of cAMP and cGMP were significantly lower than those of other two groups. Moreover, expressions of related signaling pathways including DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP were also downregulated in the corpus cavernosum of rats in aWTR group. Our finding revealed hKLK1 played a protective role in age-related ED. The DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP pathways that were linked to the mechanism hKLK1 could increase the levels of cGMP and cAMP, which might provide novel therapy targets for age-related ED.

Is the renal kallikrein-kinin system a factor that modulates hypercalciuria?

Renal tubular calcium reabsorption is one of the principal factors that determine serum calcium concentration and calcium excretion. Calcium excretion is regulated by the distal convoluted tubule and connecting tubule, where the epithelial calcium channel TRPV5 can be found, which limits the rate of transcellular calcium transport. The dynamic presence of the TRPV5 channel on the surface of the tubular cell is mediated by an endosomal recycling process. Different intrarenal factors are involved in calcium channel fixation in the apical membrane, including the anti-ageing hormone klotho and tissue kallikrein (TK). Both proteins are synthesised in the distal tubule and secreted in the tubular fluid. TK stimulates active calcium reabsorption through the bradykinin receptor B2 that compromises TRPV5 activation through the protein kinase C pathway. TK-deficient mice show hypercalciuria of renal origin comparable to that seen in TRPV5 knockout mice. There is a polymorphism with loss of function of the human TK gene R53H (allele H) that causes a marked decrease in enzymatic activity. The presence of the allele H seems to be common at least in the Japanese population (24%). These individuals have a tendency to greater calcium and sodium excretion in urine that is more evident during furosemide infusion. Future studies should analyse if manipulating the renal kallikrein-kinin system can correct idiopathic hypercalciuria with drugs other than thiazide diuretics.

132 Reduce Corporal Fibrosis to Protect Erectile Function by Inhibiting the Smad and Non-smad Pathway in the Aged Transgenic Rat Harboring Human Tissue Kallikrein 1

132 Reduce Corporal Fibrosis to Protect Erectile Function by Inhibiting the Smad and Non-smad Pathway in the Aged Transgenic Rat Harboring Human Tissue Kallikrein 1

Discovery of a new isomannide-based peptidomimetic synthetized by Ugi multicomponent reaction as human tissue kallikrein 1 inhibitor

Human kallikrein 1 (KLK1) is the most extensively studied member of this family and plays a major role in inflammation processes. From Ugi multicomponent reactions, isomannide-based peptidomimetic 10 and 13 where synthesized and showed low micromolar values of IC50 for KLK1 The most active compound (10) presented competitive mechanism, with three structural modifications important to interact with active site residues which corroborates its KLK1 inhibition. Finally, the most active compound also showed good ADMET profile, which indicates compound 10 as a potential hit in the search for new KLK1 inhibitors with low side effects.

Reduced corporal fibrosis to protect erectile function by inhibiting the Rho-kinase/LIM-kinase/cofilin pathway in the aged transgenic rat harboring human tissue kallikrein 1.

Our previous studies have demonstrated that erectile function was preserved in aged transgenic rats (TGR) harboring the human tissue kallikrein 1 (hKLK1), while the molecular level of hKLK1 on corporal fibrosis to inhibit age-related erectile dysfunction (ED) is poorly understood. Male wild-type Sprague-Dawley rats (WTR) and TGR harboring the hKLK1 gene were fed to 4- or 18-month-old and divided into three groups: young WTR (yWTR) as the control, aged WTR (aWTR), and aged TGR (aTGR). Erectile function of all rats was assessed by cavernous nerve electrostimulation method. Masson's trichrome staining was used to evaluate corporal fibrosis in the corpus cavernosum. We found that the erectile function of rats in the aWTR group was significantly lower than that of other two groups. Masson's trichrome staining revealed that compared with those of the yWTR and aTGR groups, the ratio of smooth muscle cell (SMC)/collagen (C) was significantly lower in the aWTR group. Immunohistochemistry and Western blotting analysis were performed, and results demonstrated that expression of α-SMA was lower, while expressions of transforming growth factor-β 1 (TGF-β1), RhoA, ROCK1, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group compared with those in other two groups. However, LIMK2 and cofilin expressions did not differ among three groups. Taken together, these results indicated that the RhoA/ROCK1/LIMK/cofilin pathway may be involved in the corporal fibrosis caused by advanced age, and hKLK1 may reduce this corporal fibrosis by inhibiting the activation of this pathway to ameliorate age-related ED.

Invited commentary

The purpose of this experimental animal study was to test the hypothesis that a 14-day course of intravenous human urinary kininogenase (HUK, 0.02 paranitroaniline unit) would attenuate balloon-injured carotid neointimal formation in New Zealand White rabbits fed a high-fat diet. The results were strongly positive: treatment with intravenous HUK inhibited the formation of arterial neointima by >50%, with accompanying reductions in macrophage infiltration, tumor necrosis factor-α, interleukin-1β, and transforming growth factor-β1 message and expression of transforming growth factor-β1 and p-Smad2/3 protein. So convinced were the investigators that they even demonstrated the effect angiographically after 2 months, a rarity for animal studies of this kind. The search for a safe and effective systemic method for the inhibition of neointimal proliferation after vascular or endovascular intervention has been both arduous and futile. Although significant strides have been made in controlling thrombotic and access complications, and drug-eluting stents and balloons inhibit restenosis on a local level, no systemic pharmacologic therapy has yet been shown to safely and effectively attenuate vascular proliferation. Many classes of agents have been tested, including lipid lowering drugs, antihypertensives, antiproliferatives, antiflammatories, antithrombotics, and antioxidants, but to date, none have consistently proven useful in the clinical arena. HUK represents a potentially new agent for the prevention of restenosis after vascular intervention. Made from fresh human urine, it is a tissue kallikrein with profound effects on multiple cellular processes such as inflammation, cytokine release, cellular proliferation, and membrane stabilization. Intravenous treatment with HUK has been found to reduce neurologic impairment and improve long-term outcomes after stroke; in fact, it has recently been approved in China for this indication using a dose similar to that used in this experimental study. Hopefully, the clinical safety profile of HUK will ultimately prove mild enough to also be considered for the postprocedural prevention of arterial restenosis. Human urine kininogenase attenuates balloon-induced intimal hyperplasia in rabbit carotid artery through transforming growth factor β1/Smad2/3 signaling pathwayJournal of Vascular SurgeryVol. 64Issue 4PreviewEffective treatments against restenosis after percutaneous transluminal angioplasty and stenting are largely lacking. Human tissue kallikrein gene transfer has been shown to be able to attenuate neointima formation induced by balloon catheter. As a tissue kallikrein in vivo, human urinary kininogenase (HUK) is widely used to prevent ischemia-reperfusion injury. However, the effects of HUK on neointima formation have not been explored. We therefore investigated whether HUK could alleviate balloon catheter-induced intimal hyperplasia in rabbits fed with high-fat diets. Full-Text PDF Open Archive

Preserved Erectile Function in the Aged Transgenic Rat Harboring Human Tissue Kallikrein 1

IntroductionHuman tissue kallikrein 1 (hKLK1) has enormous potential for the protection of vasodilation and endothelial function in the cardiovascular system. Our previous study proved the decreased expression of kallikrein 1 in the corpus cavernosum (CC) of aged rats, but the role of kallikrein 1 in age-related erectile dysfunction remains unknown. AimTo explore the effect and underlying mechanisms of hKLK1 on age-related erectile dysfunction. MethodsMale wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 4 and 27 months of age, respectively, and divided into four groups: young WTR (yWTR) as the control, young TGR (yTGR), aged WTR (aWTR), and aged TGR (aTGR). Rats' erectile function was evaluated by the cavernous nerve electrostimulation method. Then, CCs were collected for verification of hKLK1 followed by measurement of nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) and RhoA-Rho-kinase (ROCK) signaling activities. Masson trichrome staining and terminal deoxynucleotidyl transferase 2′-deoxyuridine 5′-triphosphate nick end labeling assay were conducted to evaluate penile fibrosis and apoptosis. Main Outcome MeasuresErectile response, NO-cGMP and RhoA-ROCK pathway-related indices, ratio of smooth muscle to collagen, and apoptosis index. ResultsThe hKLK1 alleviated the decrease of erectile function in the aWTR group. Endothelial NO synthase (eNOS) and phospho-eNOS(Ser1177) expressions, NO synthase activity, and NO and cGMP levels were decreased, whereas phospho-eNOS(Thr495), L-type Ca2+ channel, RhoA, ROCK1, ROCK2, and transforming growth factor β1 proteins were increased in the CCs of the aWTR group compared with the control yWTR group. These changes were obviously mitigated in the aTGR group. Moreover, hKLK1 prevented the sharp decrease of the ratio of smooth muscle to collagen and the increase of the apoptosis index in the CCs of the aWTR group. ConclusionThese results suggest that hKLK1 could play a preventive role in age-related erectile dysfunction by activation of the NO-cGMP pathway and inhibition of the RhoA-ROCK pathway and by antitissue fibrotic and apoptotic effects.

¿Es el sistema calicreína/quinina renal un factor modulador de la calciuria?

Renal tubular calcium reabsorption is one of the principal factors that determine serum calcium concentration and calcium excretion. Calcium excretion is regulated by the distal convoluted tubule and connecting tubule, where the epithelial calcium channel TRPV5 can be found, which limits the rate of transcellular calcium transport. The dynamic presence of the TRPV5 channel on the surface of the tubular cell is mediated by an endosomal recycling process. Different intrarenal factors are involved in calcium channel fixation in the apical membrane, including the anti-ageing hormone klotho and tissue kallikrein (TK). Both proteins are synthesised in the distal tubule and secreted in the tubular fluid. TK stimulates active calcium reabsorption through the bradykinin receptor B2 that compromises TRPV5 activation through the protein kinase C pathway. TK-deficient mice show hypercalciuria of renal origin comparable to that seen in TRPV5 knockout mice. There is a polymorphism with loss of function of the human TK gene R53H (allele H) that causes a marked decrease in enzymatic activity. The presence of the allele H seems to be common at least in the Japanese population (24%). These individuals have a tendency to greater calcium and sodium excretion in urine that is more evident during furosemide infusion. Future studies should analyse if manipulating the renal kallikrein-kinin system can correct idiopathic hypercalciuria with drugs other than thiazide diuretics.

Protective Role of Kallistatin in Vascular and Organ Injury.

Kallistatin is an endogenous protein that exerts pleiotropic effects, including vasodilation and inhibition of angiogenesis, inflammation, oxidative stress, apoptosis, fibrosis, and tumor progression. Through its 2 functional domains—an active site and a heparin-binding site—kallistatin regulates differential signaling pathways and a wide spectrum of biological functions. Kallistatin’s active site is key for inhibiting tissue kallikrein activity and stimulating the expression of endothelial nitric oxide (NO) synthase (eNOS), sirtuin 1 (SIRT1), and suppressor of cytokine signaling 3 (SOCS3). Kallistatin via its heparin-binding site blocks signaling pathways mediated by growth factors and cytokines, such as vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), high-mobility group box-1 (HMGB1), Wnt, transforming growth factor-β (TGF-β), and epidermal growth factor. Kallistatin gene or protein delivery protects against the pathogenesis of hypertension, heart and kidney damage, arthritis, sepsis, influenza virus infection, tumor growth, and metastasis in animal models. Conversely, depletion of endogenous kallistatin by neutralizing antibody injection exacerbates cardiovascular and renal injury in hypertensive rats. Kallistatin levels are markedly reduced in rodents with hypertension, sepsis, streptozotocin-induced diabetes mellitus, and cardiac and renal injury. Kallistatin levels are also diminished in patients with liver disease, septic syndrome, diabetic retinopathy, severe pneumonia, inflammatory bowel disease, obesity, and prostate, and colon cancer. Therefore, circulating kallistatin levels may serve as a new biomarker for human diseases. This review summarizes kallistatin’s protective roles and mechanisms in vascular and organ injury and highlights the therapeutic potential of kallistatin for multiple disease states. Kallistatin was discovered in human plasma as a tissue kallikrein–binding protein.1 Sequence analysis indicated that tissue kallikrein–binding protein is a novel serine proteinase inhibitor (serpin) and was later designated kallistatin because of its ability to inhibit tissue kallikrein activity.2–9 Tissue kallikrein is a serine proteinase that cleaves low–molecular weight kininogen substrate to release vasoactive kinin peptides.10 The …

Co-overexpression of human tissue kallikrein 1 and human metalloproteinase 1 tissue inhibitor inhibits neointima formation in the rat artery after balloon angioplasty

To investigate the effects of adenovirus-mediated human tissue kallikrein 1(hTK-1) and/or human tissue metalloproteinase inhibitor 1 (hTIMP-1) gene delivery on the neointima formation in balloon-injured rat carotids and related mechanism. Forty-six male Sprague-Dawley rats were randomly assigned into 6 groups with the random number table: (1) sham-operated group(n=6), (2) angioplasty group (n=8), (3) vector virus group (n=8), (4) hTK-1 group (n=8), (5) hTIMP-1 group (n=8), (6) hTK-1-hTIMP-1 group (n=8). Except sham rats, all rats underwent carotid artery balloon injury and local delivery of saline or different recombined adenoviruses respectively. Rats were sacrificed 14 days later. Intima/media area ratio was assessed on hematoxylin-eosin stained tissue section. Immunofluorescence images stained for hTK-1, hTIMP-1 were obtained and analyzed by the confocal microscope for co-localization examination of hTK-1 and hTIMP-1. The protein expression levels of hTK-1, hTIMP-1, matrix metalloproteinases(MMP)-2 and MMP-9 were determined by Western blot. Immune histochemical staining for PCNA was also performed. (1)Intima area, intima/media area ratio, PCNA, MMP-2 and MMP-9 levels were all significantly increased in rats underwent angioplasty (did or did not receive vector virus) compared with sham-operated rats (all P<0.01) while above parameters were similar between rats underwent angioplasty or vector virus delivery (all P>0.05). (2) The intima area of rats received vector virus, hTK-1, hTIMP-1 or dual gene transfer were (0.160±0.010), (0.110±0.015), (0.121±0.016) or (0.081±0.008) mm(2) respectively, intima area was similar between rats received hTK-1 or hTIMP-1 (P>0.05), differences were found between other groups (all P<0.01). The intima/media area ratio of rats received vector virus, hTK-1, hTIMP-1 or dual gene transfer were 2.035±0.117, 1.443±0.097, 1.522±0.078 or 0.972±0.072 respectively, no difference was found between rats received hTK-1 or hTIMP-1 in intima/media area ratio (all P>0.05), differences were found between other groups (all P<0.01). The MMP-2 and MMP-9 expression of rats received vector virus, hTK-1, hTIMP-1 or dual gene transfer were 0.817±0.036, 0.606±0.044, 0.571±0.061 or 0.455±0.030 and 0.745±0.057, 0.613±0.038, 0.582±0.050 or 0.473±0.038 respectively, no difference was found between rats received hTK-1 or hTIMP-1 in MMP-2 or MMP-9 expression (all P>0.05), differences were found between other groups (all P<0.01). The PCNA expression of rats received vector virus, hTK-1, hTIMP-1 or dual gene transfer were 0.065±0.007, 0.052±0.004, 0.055±0.007 or 0.031±0.004 respectively, no difference was found between rats received hTK-1or hTIMP-1 in PCNA expression (all P>0.05), differences were found between other groups (all P<0.01). hTK-1 and hTIMP-1 co-overexpression may synergistically inhibit neointimal hyperplasia, attenuate vascular remodeling and reduce restenosis possibly via down regulating the expressions of PCNA, MMP-2 and MMP-9 in balloon-injured rat carotids.

The isolated human umbilical vein as a bioassay for kinin-generating proteases: An in vitro model for therapeutic angioedema agents

AimsThe isolated human umbilical vein is a robust contractile bioassay for ligands of the bradykinin (BK) B2 receptor (B2R), also extendable to B1 receptor (B1R) pharmacology. We hypothesized that, as a freshly isolated vessel, it also contains traces of plasma proteins that may confer responses to exogenous proteases via the formation of kinins. Main methodsRings of human umbilical veins were mounted in organ baths containing Krebs buffer maintained at 37°C and purified proteases were introduced in the bathing fluid along with additional drugs/proteins that permit mechanistic analysis of effects. Key findingsThe previously described contractile response to human recombinant tissue kallikrein (KLK-1, 1–10nM) is not influenced by metabolic inhibitors, suggesting its dependence on a preexisting reservoir of low molecular weight-kininogen (LK). Active plasma kallikrein (apK, ≤5nM) was inactive in fresh tissues, unless high molecular weight-kininogen (HK, 39–197nM) replenishment was applied. The effects of KLK-1 and HK+apK are abolished by pretreating tissues with icatibant, but not with tranexamic acid. C1-esterase inhibitor inhibited only HK+apK. Purified plasmin and neutrophil proteinase-3 produced small contractions in the presence of HK only, and tissue plasminogen activator, none. B1R stimulation was pharmacologically evidenced in response to KLK-1 if LK was supplied. SignificanceThe pharmacology of KLK-1 and HK+apK in the human isolated umbilical vein is essentially based on the activity of locally generated kinins and this assay models the inhibitory action of some therapeutic agents active in angioedema states. Proteases that indirectly generate kinins have little activity in the system.

MP86-12 HUMAN TISSUE KALLIKREIN 1 RESTORES ERECTILE FUNCTION OF STREPTOZOTOCIN- INDUCED DIABETIC RATS BY ACTIVATING PI3K/ENOS PATHWAY, INHIBITING OXIDATIVE STRESS AND APOPTOSIS IN THE CORPUS CAVERNOSUM

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology I1 Apr 2016MP86-12 HUMAN TISSUE KALLIKREIN 1 RESTORES ERECTILE FUNCTION OF STREPTOZOTOCIN- INDUCED DIABETIC RATS BY ACTIVATING PI3K/ENOS PATHWAY, INHIBITING OXIDATIVE STRESS AND APOPTOSIS IN THE CORPUS CAVERNOSUM Yang Luan, Yajun Ruan, Tao Wang, Yan Zhang, Kai Cui, Rui Li, Jun Yang, Ruibao Chen, Shaogang Wang, Jihong Liu, and Zhangqun Ye Yang LuanYang Luan More articles by this author , Yajun RuanYajun Ruan More articles by this author , Tao WangTao Wang More articles by this author , Yan ZhangYan Zhang More articles by this author , Kai CuiKai Cui More articles by this author , Rui LiRui Li More articles by this author , Jun YangJun Yang More articles by this author , Ruibao ChenRuibao Chen More articles by this author , Shaogang WangShaogang Wang More articles by this author , Jihong LiuJihong Liu More articles by this author , and Zhangqun YeZhangqun Ye More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2320AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our previous study has proved the protective role of human tissue kallikrein 1 (hKLK1) in erectile function of aged rats with the transgenic Sprague-Dawley (SD) rats harboring hKLK1 gene (TGR), but its effect on diabetic erectile dysfunction (ED) is still unknown. METHODS Eight-week-old male wild type SD rats (WTR) and age matched TGR were randomly divided into five groups: citrate buffer treated (1) WTR and (2) TGR; streptozotocin-induced (3) diabetic wild type rats (WTDM) and (4) diabetic transgenic rats (TGDM); (5) TGDM with daily administration of bradykinin receptor 2 inhibitor HOE 140 (TGDMH). Erectile response was measured by cavernous nerve electrostimulation after 12-week treatment. Then the corpus cavernosum (CC) tissues were harvested for hKLK1 gene identification and mechanism exploration. The regulation of hKLK1 on PI 3 kinase (PI3K)/ endothelial nitric oxide synthase (eNOS) pathway was detected within CC in vivo and TGR derived cavernous smooth muscle cell (CSMC) in vitro. In addition, the effect of hKLK1 on oxidative stress and apoptosis in CC of diabetic rats and high glucose treated CSMC was also evaluated. RESULTS hKLK1 attenuated the decreased erectile function of WTDM, while the protective effect was abolished in TGDMH. The decrease of PI3K(p55), AKT, eNOS, phospho-eNOS(Ser1177), cyclic guanosine monophosphate (cGMP) and increase of phospho-eNOS(Thr495), phosphodiesterase type 5, L-type calcium channel in CC of WTDM were improved in that of TGDM, but deteriorated again by HOE 140. Meanwhile, hKLK1 could elevate the level of cGMP and reduce the concentration of Ca2+ in CSMC in vitro, but these changes were diminished by B2R, PI3K or eNOS inhibitors. In addition, the CC of WTDM had higher proteins expression of nicotinamide adenine dinucleotide phosphate oxidase, malondialdehyde level, lower activity of superoxide dismutase and elevated apoptosis percentage when compared to WTR. However, hKLK1 improved these parameters in TGDM and inhibition with HOE 140 would abolish these advantages. Besides, the level of reactive oxygen species (ROS) and apoptosis rate elevated in high glucose treated CSMC in a concentration-dependent manner and hKLK1 could considerably inhibit the generation of ROS and cell apoptosis in vitro. CONCLUSIONS These results indicate that hKLK1 could restore diabetic ED through activation of PI3K/eNOS signaling and inhibition of oxidative stress and apoptosis in CC. © 2016FiguresReferencesRelatedDetailsCited bySeftel A (2016) Re: Preserved Erectile Function in the Aged Transgenic Rat Harboring Human Tissue Kallikrein 1Journal of Urology, VOL. 197, NO. 2, (493-493), Online publication date: 1-Feb-2017. Volume 195Issue 4SApril 2016Page: e1110 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yang Luan More articles by this author Yajun Ruan More articles by this author Tao Wang More articles by this author Yan Zhang More articles by this author Kai Cui More articles by this author Rui Li More articles by this author Jun Yang More articles by this author Ruibao Chen More articles by this author Shaogang Wang More articles by this author Jihong Liu More articles by this author Zhangqun Ye More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Synthesis, biological evaluation and molecular modeling of pseudo-peptides based statine as inhibitors for human tissue kallikrein 5

Human kallikrein 5 (KLK5) is a potential target for the treatment of skin inflammation and cancer. A new series of statine based peptidomimetic compounds were designed and synthesized through simple and efficient reactions. Some KLK5 inhibitors (2a–c compounds) were identified with nanomolar affinity showing Ki values of 0.12-0.13 μM. Our molecular modeling studies suggest that the inhibitors binding at the KLK5 through H-bond interactions with key residues (mainly His108, Gln242, Gly243, Ser245, and Ser260), disrupting the correlated motions mainly among the Ile67-Tyr127, Glu128-Val187, and Gly237-Ser293 subdomains, which seems to be crucial for KLK5 activity. Therefore, we believe that these findings will significantly facilitate our understanding of the conformational dynamics in the course of KLK5 inhibition and, consequently, the development of more potent molecules as alternative for cancer treatment.