Recent advances in the discovery and development of drugs targeting the kallikrein-kinin system.

BACKGROUND: The kallikrein-kinin system is a key regulatory cascade involved in blood pressure maintenance, hemostasis, inflammation and renal function. Currently, approved drugs remain limited to the rare disease hereditary angioedema. However, growing interest in this system is indicated by an increasing number of promising drug candidates for further indications. METHODS: To provide an overview of current drug development, a two-stage literature search was conducted between March and December 2023 to identify drug candidates with targets in the kallikrein-kinin system. First, drug candidates were identified using PubMed and Clinicaltrials.gov. Second, the latest publications/results for these compounds were searched in PubMed, Clinicaltrials.gov and Google Scholar. The findings were categorized by target, stage of development, and intended indication. RESULTS: The search identified 68 drugs, of which 10 are approved, 25 are in clinical development, and 33 in preclinical development. The three most studied indications included diabetic retinopathy, thromboprophylaxis and hereditary angioedema. The latter is still an indication for most of the drug candidates close to regulatory approval (3 out of 4). For the emerging indications, promising new drug candidates in clinical development are ixodes ricinus-contact phase inhibitor for thromboprophylaxis and RZ402 and THR-149 for the treatment of diabetic macular edema (all phase 2). CONCLUSION: The therapeutic impact of targeting the kallikrein-kinin system is no longer limited to the treatment of hereditary angioedema. Ongoing research on other diseases demonstrates the potential of therapeutic interventions targeting the kallikrein-kinin system and will provide further treatment options for patients in the future.

hKLK alleviates myocardial fibrosis in mice with viral myocarditis.

Myocardial fibrosis is the most serious complication of viral myocarditis (VMC). This study aimed to investigate the therapeutic benefits and underlying mechanisms of lentivirus-mediated human tissue kallikrein gene transfer in myocardial fibrosis in VMC mice. We established VMC mouse model via intraperitoneal injection with Coxsackie B3 virus. The effect was then assessed after treatment with vehicle, the empty lentiviral vectors (EZ.null), and the vectors expressing hKLK1 (EZ.hKLK1) via tail vein injection for 30 days, respectively. The results showed that administering EZ.hKLK1 successfully induced hKLK1 overexpression in mouse heart. Compared with EZ.null treatment, EZ.hKLK1 administration significantly reduced the heart/weight ratio, improved cardiac function, and ameliorated myocardial inflammation in VMC mice, suggesting that hKLK1 overexpression alleviates VMC in mice. EZ.hKLK1 administration also significantly abrogated the increased myocardial collagen content, type I/III collagen ratio, TGF-ß1 mRNA and protein expression in VMC mice, suggesting that hKLK1 overexpression reduces collagen accumulation and blunts TGF-ß1 signaling in the hearts of VMC mice. In conclusion, our results suggest that hKLK1 alleviates myocardial fibrosis in VMC mice, possibly by downregulating TGF-ß1 expression.

Decreased Tissue Kallikrein Levels and the Risk of Ischemic Stroke: A Community-Based Cross-Sectional Study in China.

AIM: Tissue kallikrein (TK) exerts protective effects on cardiac cerebrovascular diseases (CCVDs). Changes in TK level in plasma are associated with ischemic stroke and coronary artery disease (CAD); however, a causal correlation could not be established. Therefore, we investigated the association between TK levels and CCVDs in a community-based cross-sectional study in China. METHODS: A total of 6043 subjects (4242 men and 1801 women) were enrolled in this community-based cross-sectional study. Then, TK levels were measured using an enzyme-linked immunosorbent assay kit. Multivariate linear regression model and logistic regression were used to assess the correlations between TK levels and CCVDs. Subsequently, the receiver operating characteristic (ROC) curve was drawn to assess the value of TK level in evaluating the risk of ischemic stroke. Finally, the influence of various medications was evaluated on TK levels. RESULTS: The TK level was significantly lower in subjects with ischemic stroke (P < 0.001) and hypertension (P < 0.001) and negatively associated with ischemic stroke (P < 0.001) but not associated with hypertension, coronary heart disease, and diabetes compared to the traditional risk factors. The diagnostic accuracy for ischemic stroke, as quantified by the area under the curve, was 0.892 (95% CI, 0.884-0.900) for TK level, deeming it as a promising assessment tool. Moreover, no appreciable influence of various drugs therapy was found in TK levels (P = 0.222) except for those taking antilipemic agents. CONCLUSION: TK is a strong and independent endogenous protective factor against ischemic stroke in the Chinese population and could be a promising biomarker for the risk of ischemic stroke.

Relationship between serum KLK11 and MK levels and the efficacy of the first 131I ablation after surgery for differentiated thyroid cancer / 中华内分泌外科杂志

Objective:To explore and analyze the relationship between serum KLK11 and MK levels and the effect of first iodine 131 ( 131I) ablation after operation for differentiated thyroid cancer. Method:108 patients with differentiated thyroid cancer who underwent total thyroidectomy in our hospital from Jun. 2020 to Jun. 2021 were consecutively selected, and received radioactive ablation after surgery. There were 37 males and 71 females. The age was (48.32±4.25) years, ranging from 28 to 79 years. The patients were divided into successful ablation according to whether the ablation was successful after treatment. There were 64 cases in the group and 44 cases in the unsuccessful group, and 60 healthy people with no abnormality in physical examination during the same period were selected as the control group. The patients were divided into a metastasis-positive group of 20 cases and a metastasis-negative group of 88 cases according to whether lymph nodes occurred. After surgery, serum samples of all subjects were taken, and enzyme-linked immunosorbent assay was used to detect the levels of serum kallikrein-related peptidases 11 (KLK11) and midkine (MK) , and the levels of serum KLK11 and MK were analyzed. Gender, age, BMI, TNM stage, TSH, maximum diameter of lesion, and duration of nail removal were collected. Univariate analysis and logistic regression analysis were used to analyze the independent risk factors of postoperative efficacy.Result:The levels of serum KLK11 and MK in the successful and unsuccessful groups were higher than those in the control group, while the levels of KLK11 and MK in the unsuccessful group were higher than those in the successful group (KLK11: t= 2.642, P<0.05; MK: t=11.906, P<0.05) . The serum levels of KLK11 and MK in the metastasis-positive group were higher than those in the metastasis-negative group (KLK11: t= 2.908, P<0.05; MK: t=14.907, P<0.05) . Univariate analysis showed that BMI ( χ2=6.780, P=0.009) , maximum diameter of lesions ( χ2=14.819, P=0.001) , TSH ( χ2=13.627, P=0.001) , serum KLK11 ( t=2.642, P=0.01) , and serum MK ( t=11.906, P<0.001) were associated with the effect of first 131I ablation after surgery for differentiated thyroid cancer ( P<0.05) . Taking the success of ablation as the dependent variable, a multivariate logistic regression analysis was performed. The results showed that the maximum diameter of the lesions greater than 2 cm ( OR=10.740, 95%CI: 7.033-16.401) , increased level of TSH ( OR=8.559, 95%CI: 2.812-26.057) , increased serum KLK11 level ( OR=16.710, 95%CI: .548-32.666) and increased serum MK level ( OR=10.580, 95%CI: 6.294-17.786) were the factors affecting the first 131I ablation effect after DTC surgery ( P<0.05) . Conclusion:The elevated levels of serum KLK11 and MK are independent risk factors affecting the efficacy of the first 131I ablation after surgery for differentiated thyroid cancer.

Increase in Blood-Brain Barrier Permeability is Modulated by Tissue Kallikrein via Activation of Bradykinin B1 and B2 Receptor-Mediated Signaling.

AIM: Disruption of the blood-brain barrier (BBB) is a critical pathological feature after stroke. Although tissue kallikrein (TK) has used in the treatment of stroke in China, the role of TK in modulating BBB permeability is not clear. METHODS: We investigated the effect of different doses of TK on BBB by in vivo assessments of Evans blue (EB) and sodium-fluorescein isothiocyanate (FITC) leakage and in vitro assessments of the integrity of BBB and monolayers of microvascular endothelial cells (BMVECs). The expression of zonula occludens-1 (ZO-1) and bradykinin receptor-mediated signaling in BMVECs was detected. RESULTS: A significant increase in BBB permeability was observed in the mice treated with high dose of TK. However, standard and medium doses of TK could only enable sodium-FITC to enter the brain. The result of in vitro study indicated that high-doses of TK, but not standard and medium-dose of TK, reduced normal BBB integrity accompanied by a decreased expression of zonula occludens-1 (ZO-1), upregulated the mRNA levels of bradykinin 2 receptor (B2R) and endothelial nitric oxide synthase (eNOS) and the abundance of B2R. Moreover, standard-dose of TK exacerbated lipopolysaccharide-induced BBB hyperpermeability, upregulated the mRNA levels of bradykinin 1 receptor (B1R) and inducible nitric oxide synthase (iNOS), increased the abundance of B1R and reduced the abundance of ZO-1; these effects were inhibited by TK inhibitor. CONCLUSION: TK can disrupt tight junctions and increase normal BBB permeability via B2R-dependent eNOS signaling pathway, aggravate impairment of BBB via B1R-dependent iNOS signaling pathway, and consequently serve as a useful adjunctive treatment for enhancing the efficacy of other neurotherapeutics.

Tissue Kallikrein Exacerbating Sepsis-Induced Endothelial Hyperpermeability is Highly Predictive of Severity and Mortality in Sepsis.

AIM: Sepsis, an acute, life-threatening dysregulated response to infection, affects practically all aspects of endothelial function. Tissue kallikrein (TK) is a key enzyme in the kallikrein-kinin system (KKS) which has been implicated in endothelial permeability. Thus, we aimed to establish a potentially novel association among TK, endothelial permeability, and sepsis demonstrated by clinical investigation and in vitro studies. METHODS: We performed a clinical investigation with the participation of a total of 76 controls, 42 systemic inflammatory response syndrome (SIRS) patients, and 150 patients with sepsis, who were followed-up for 28 days. Circulating TK levels were measured with an enzyme-linked immunosorbent assay. Then, the effect of TK on sepsis-induced endothelial hyperpermeability was evaluated by in vitro study. RESULTS: Data showed a gradual increase in TK level among controls and the patients with SIRS, sepsis, and septic shock (0.288±0.097 mg/l vs 0.335±0.149 vs 0.495±0.170 vs 0.531±0.188 mg/l, respectively, P <0.001). Further analysis revealed that plasma TK level was positively associated with the severity and mortality of sepsis and negatively associated with event-free survival during 28 days of follow-up (relative risk, 3.333; 95% CI, 2.255-4.925; p < 0.001). With a septic model of TK and kallistatin in vitro, we found that TK exacerbated sepsis-induced endothelial hyperpermeability by downregulating zonula occluden-1 (ZO-1) and vascular endothelial (VE)-cadherin, and these could be reversed by kallistatin, an inhibitor of TK. CONCLUSION: TK can be used in the diagnosis of sepsis and assessment of severity and prognosis of disease. Inhibition of TK may be a novel therapeutic target for sepsis through increasing ZO-1 and VE-cadherin, as well as downregulating endothelial permeability.

Co­expression of tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 improves myocardial ischemia­reperfusion injury by promoting angiogenesis and inhibiting oxidative stress.

Myocardial ischemia/reperfusion (I/R) injury is a serious complication of reperfusion therapy for myocardial infarction. At present, there is not an effective treatment strategy available for myocardial I/R. The present study aimed to investigate the effects of human tissue kallikrein 1 (hTK1) and human tissue inhibitors of matrix metalloproteinase 1 (hTIMP1) gene co­expression on myocardial I/R injury. A rat model of myocardial I/R injury and a cell model with hypoxia/reoxygenation (H/R) treatment in cardiac microvascular endothelial cells (CMVECs) were established, and treated with adenovirus (Ad)­hTK1/hTIMP1. Following which, histological and triphenyl­tetrazolium­chloride staining assays were performed. Cardiac function was tested by echocardiographic measurement. The serum levels of oxidative stress biomarkers in rats and the intracellular reactive oxygen species (ROS) levels in CMVECs were measured. Additionally, experiments, including immunostaining, reverse transcription­quantitative PCR, western blotting, and MTT, wound healing, Transwell and tube formation assays were also performed. The results of the present study demonstrated that Ad­hTK1/hTIMP1 alleviated myocardial injury and improved cardiac function in myocardial I/R model rats. Ad­hTK1/hTIMP1 also significantly enhanced microvessel formation, decreased matrix metalloproteinase (MMP)2 and MMP9 expression, and reduced oxidative stress in myocardial I/R model rats. Furthermore, Ad­hTK1/hTIMP1 significantly enhanced proliferation, migration and tube formation in H/R­treated CMVECs. Additionally, Ad­hTK1/hTIMP1 significantly decreased intracellular ROS production and γ­H2A.X variant histone expression levels in H/R­treated CMVECs. In conclusion, the results of the present study demonstrated that co­expression of hTK1 and hTIMP1 genes displayed significant protective effects on myocardial I/R injury by promoting angiogenesis and suppressing oxidative stress; therefore, co­expression of hTK1 and hTIMP1 may serve as a potential therapeutic strategy for myocardial I/R injury.

Heparin Blocks the Inhibition of Tissue Kallikrein 1 by Kallistatin through Electrostatic Repulsion.

Kallistatin, also known as SERPINA4, has been implicated in the regulation of blood pressure and angiogenesis, due to its specific inhibition of tissue kallikrein 1 (KLK1) and/or by its heparin binding ability. The binding of heparin on kallistatin has been shown to block the inhibition of KLK1 by kallistatin but the detailed molecular mechanism underlying this blockade is unclear. Here we solved the crystal structures of human kallistatin and its complex with heparin at 1.9 and 1.8 Å resolution, respectively. The structures show that kallistatin has a conserved serpin fold and undergoes typical stressed-to-relaxed conformational changes upon reactive loop cleavage. Structural analysis and mutagenesis studies show that the heparin binding site of kallistatin is located on a surface with positive electrostatic potential near a unique protruded 310 helix between helix H and strand 2 of ß-sheet C. Heparin binding on this site would prevent KLK1 from docking onto kallistatin due to the electrostatic repulsion between heparin and the negatively charged surface of KLK1, thus blocking the inhibition of KLK1 by kallistatin. Replacement of the acidic exosite 1 residues of KLK1 with basic amino acids as in thrombin resulted in accelerated inhibition. Taken together, these data indicate that heparin controls the specificity of kallistatin, such that kinin generation by KLK1 within the microcirculation will be locally protected by the binding of kallistatin to the heparin-like glycosaminoglycans of the endothelium.

Kinin B1 Receptor Signaling in Skin Homeostasis and Wound Healing.

Kinins are proinflammatory peptides that are formed in the skin by the enzymatic action of tissue kallikrein (KLK1) on kininogens. Tissue kallikrein is produced by eccrine sweat glands and also by cells of the stratum granulosum and other skin appendages. Kinin formation may be favored during inflammatory skin disorders when plasma constituents, including kininogens, extravasate from venules and capillaries, which have increased permeability in response to the plethora of inflammatory mediators generated in the course of acute inflammation. By activating either kinin B1 or B2 receptors, kinins modulate keratinocyte differentiation, which relays on activation of several signaling systems that follows receptor stimulation. Participation of the kinin B1 receptor in wound healing is still a matter of controversy though some studies indicate that B1 receptor stimulation regulates keratinocyte migration by controlling metalloproteases 2 and 9 production and by improving wound closure in a mouse model. Development of more stable kinin B1 receptor agonists may be beneficial to modulate wound healing, especially if we take into account that the B1 receptor is up-regulated by inflammation and by cytokines generated in the inflamed microenvironment.

Neuroprotective mechanism of TMP269, a selective class IIA histone deacetylase inhibitor, after cerebral ischemia/reperfusion injury.

TMP269 is a selective class IIA histone deacetylase inhibitor that has a protective effect on the central nervous system, whose specific mechanism of action is unclear. We aimed to reveal the optimal concentration of TMP269 for protecting against cerebral ischemia/reperfusion injury and its neuroprotective mechanism. Male Sprague-Dawley rats were randomly divided into sham, ischemia/reperfusion, and 1, 4, 10 and 16 mg/kg TMP269 groups. Cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion. TMP269 was intraperitoneally administered at different doses 0.5 hours before ischemia induction. Western blot assay and immunohistochemistry were used to detect effects of TMP269 on histone 2 acetylation. The results showed that the level of histone 2 acetylation was increased 24 hours after TMP269 injection. 2,3,5-Triphenyltetrazolium chloride staining was utilized to examine effect of TMP269 on infarct volume. The results found that different doses of TMP269 could reduce the infarct volume. Western blot assay, immunohistochemistry and Evans blue staining were employed to measure the effect of TMP269 on blood-brain barrier. The results showed that TMP269 counteracted the abnormal endothelial cell permeability changes caused by cerebral ischemia/reperfusion. Western blot assay and immunohistochemistry were used to determine the effect of TMP269 on tissue kallikrein. The results found that TMP269 up-regulated the expression of tissue kallikrein. Western blot assay further determined the optimal concentration to be 4 mg/kg. In conclusion, TMP269 plays a neuroprotective role by up-regulating the level of histone 2 acetylation, alleviating endothelial cell injury after cerebral ischemia/reperfusion, and up-regulating the expression of tissue kallikrein. The experimental protocol was approved in 2014 by the Department of Laboratory Animal Science, Fudan University, China (approval No. 20140143C001).

Plasma concentrations of tissue kallikrein in normal and preeclamptic pregnancies.

Objective: To investigate maternal plasma concentrations of tissue kallikrein (TK) in normal and preeclamptic pregnancies.Methods: 96 women with singleton pregnancies were categorized into normal, mild preeclampsia and preeclampsia with severe features. Plasma levels of TK were quantified by ELISA and left lateralrecumbencyposition BP measured.Results: Maternal plasma TK concentrations were significantly lower in preeclampsia with severe features compared with mild preeclampsia and normal pregnant. Plasma TK concentrations were negatively correlated with systolic and diastolic blood pressure, and 24-hour urine protein.Conclusion: Lower maternal plasma TK may be a risk marker that reflects the severity of preeclampsia.

Tissue kallikrein: A potential serum biomarker to predict delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage.

BACKGROUND: Delayed cerebral ischemia (DCI) is a severe complication after aneurysmal subarachnoid hemorrhage (aSAH). Tissue kallikrein (TK), a subgroup of serine proteinases, is an important component of the kallikrein-kinin system. Exogenous TK attenuated cerebral vasospasm in a rabbit model of subarachnoid hemorrhage. We intended to discern association of serum TK levels with aSAH-related DCI. METHODS: Serum TK levels were detected in a total of 92 aSAH patients and 92 healthy controls. A multivariate logistic regression model was configured to investigate relationship between TK levels and occurrence of DCI. RESULTS: TK levels were substantially lower in aSAH patients than in controls. TK levels were strongly correlated with World Federation of Neurological Surgeons (WFNS) score and modified Fisher score. Serum TK, WFNS score and modified Fisher score retained as the three independent predictors for DCI. Under receiver operating characteristic curve, predictive capability of TK levels was in the range of WFNS score and modified Fisher score, as well as TK levels could remarkably improve predictive abilities of WFNS score and modified Fisher score. CONCLUSIONS: Serum TK emerges as a potential biomarker for assessment of hemorrhagic severity and prediction of DCI following aSAH.

Determination of serum tissue kallikrein levels after traumatic brain injury.

BACKGROUND: Tissue kallikrein (TK) plays an important role in the kallikrein-kinin system. Its protective role has been demonstrated in traumatic brain injury (TBI). We attempted to determine relationship between serum TK levels and trauma severity in addition to clinical outcome in TBI. METHODS: We recruited 112 patients with severe TBI (Glasgow coma scale score < 9) and 112 controls. We configured 2 multivariate models to assess the relationship between serum TK levels and 30-day death. Its prognostic predictive ability was analyzed under receiver operating characteristic curve. RESULTS: TK levels were significantly lower in patients than in controls (median 0.148 mg/l, the upper - lower quartiles 0.121-0.185 vs. median 0.258 mg/l, the upper - lower quartiles 0.207-0.342, P < 0.001). TK levels were closely and positively correlated with Glasgow coma scale score (r = 0.550). TK levels <0.148 mg/l independently predicted 30-day mortality with odds ratio value of 4.752 (95% confidence interval (CI), 1.166-19.367) and 30-day overall survival with hazard ratio value of 3.698 (95% CI, 1.026-13.333). TK levels significantly discriminated 30-day mortality with area under curve of 0.822 (95% CI, 0.738-0.887). CONCLUSIONS: Serum TK can represent a potential predictor of clinical outcome in TBI patients.

Human tissue kallikrein in the treatment of acute ischemic stroke.

Acute ischemic stroke (AIS) remains a major cause of death and disability throughout the world. The most severe form of stroke results from large vessel occlusion of the major branches of the Circle of Willis. The treatment strategies currently available in western countries for large vessel occlusion involve rapid restoration of blood flow through removal of the offending blood clot using mechanical or pharmacological means (e.g. tissue plasma activator; tPA). This review assesses prospects for a novel pharmacological approach to enhance the availability of the natural enzyme tissue kallikrein (KLK1), an important regulator of local blood flow. KLK1 is responsible for the generation of kinins (bradykinin and kallidin), which promote local vasodilation and long-term vascularization. Moreover, KLK1 has been used clinically as a direct treatment for multiple diseases associated with impaired local blood flow including AIS. A form of human KLK1 isolated from human urine is approved in the People's Republic of China for subacute treatment of AIS. Here we review the rationale for using KLK1 as an additional pharmacological treatment for AIS by providing the biochemical mechanism as well as the human clinical data that support this approach.

Human tissue kallikrein-1 protects against the development of erectile dysfunction in a rat model of hyperhomocysteinemia.

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.

Human tissue kallikrein-1 protects against the development of erectile dysfunction in a rat model of hyperhomocysteinemia / 亚洲男科学杂志(英文版)

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.

Human tissue kallikrein-1 protects against the development of erectile dysfunction in a rat model of hyperhomocysteinemia / 亚洲男科学杂志(英文版)

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.

Functional interrelationships between the kallikrein-related peptidases family and the classical kinin system in the human neutrophil.

In the human neutrophil, kallikrein-related peptidases (KLKs) have a significant functional relationship with the classical kinin system as a kinin B1 receptor agonist induces secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the medium. Secretion of KLK1, the kinin-forming enzyme, may perpetuate formation of kinin in the inflammatory milieu by hydrolyzing extravasated kininogens present in tissue edema. Secretion of KLKs into the inflammatory milieu, induced by kinins or other proinflammatory mediators, provides the human neutrophil with a wide range of molecular interactions to hydrolyze different cellular and extracellular matrix components, which may be of critical relevance in different mechanisms involving inflammation.

Remote limb ischemic postconditioning promotes motor function recovery in a rat model of ischemic stroke via the up-regulation of endogenous tissue kallikrein.

AIMS: Remote ischemic conditionings, such as pre- and per-conditioning, are known to provide cardioprotection in animal models of ischemia. However, little is known about the neuroprotection effect of postconditioning after cerebral ischemia. In this study, we aim to evaluate the motor function rescuing effect of remote limb ischemic postconditioning (RIPostC) in a rat model of acute cerebral stroke. METHODS: Left middle cerebral artery occlusion (MCAO) was performed to generate the rat model of ischemic stroke, followed by daily RIPostC treatment for maximum 21 days. The motor function after RIPostC was assessed with foot fault test and balance beam test. Local infarct volume was measured through MRI scanning. Neuronal status was evaluated with Nissl's, HE, and MAP2 immunostaining. Lectin immunostaining was performed to evaluate the microvessel density and area. RESULTS: Daily RIPostC for more than 21 days promoted motor function recovery and provided long-lasting neuroprotection after MCAO. Reduced infarct volume, rescued neuronal loss, and enhanced microvessel density and size in the injured areas were observed. In addition, the RIPostC effect was associated with the up-regulation of endogenous tissue kallikrein (TK) level in circulating blood and local ischemic brain regions. A TK receptor antagonist HOE-140 partially reversed RIPostC-induced improvements, indicating the specificity of endogenous TK mediating the neuroprotection effect of RIPostC. CONCLUSION: Our study demonstrates RIPostC treatment as an effective rehabilitation therapy to provide motor function recovery and alleviate brain impairment in a rat model of acute cerebral ischemia. We also for the first time provide evidence showing that the up-regulation of endogenous TK from remote conditioning regions underlies the observed effects of RIPostC.

Kinin B1 receptors as a therapeutic target for inflammation.

INTRODUCTION: Kinins are peptide mediators exerting their pro-inflammatory actions by the selective stimulation of two distinct G-protein coupled receptors, termed BKB1R and BKB2R. While BKB2R is constitutively expressed in a multitude of tissues, BKB1R is hardly expressed at baseline but highly inducible by inflammatory mediators. In particular, BKB1R was shown to be involved in the pathogenesis of numerous inflammatory diseases. Areas covered: This review intends to evaluate the therapeutic potential of substances interacting with the BKB1R. To this purpose we summarize the published literature on animal studies with antagonists and knockout mice for this receptor. Expert Opinion: In most cases the pharmacological inhibition of BKB1R or its genetic deletion was beneficial for the outcome of the disease in animal models. Therefore, several companies have developed BKB1R antagonists and tested them in phase I and II clinical trials. However, none of the developed BKB1R antagonists was further developed for clinical use. We discuss possible reasons for this failure of translation of preclinical findings on BKB1R antagonists into the clinic.