Tissue Kallikrein Research Articles

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.

Crystal structures of the complex of a kallikrein inhibitor from Bauhinia bauhinioides with trypsin and modeling of kallikrein complexes.

Structures of a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides (BbKI) complexed with bovine trypsin were determined in two crystal forms. The crystal structure with the L55R mutant of BbKI was determined in space group P64 at 1.94 Å resolution and that with native BbKI in the monoclinic space group P21 at 3.95 Å resolution. The asymmetric unit of the latter crystals contained 44 independent complexes, thus representing one of the largest numbers of independent objects deposited in the Protein Data Bank. Additionally, the structure of the complex with native BbKI was determined at 2.0 Å resolution from P64 crystals isomorphous to those of the mutant. Since BbKI has previously been found to be a potent inhibitor of the trypsin-like plasma kallikrein, it was also tested against several tissue kallikreins. It was found that BbKI is a potent inhibitor of human tissue kallikrein 4 (KLK4) and the chymotrypsin-like human tissue kallikrein 7 (KLK7). Structures of BbKI complexed with the catalytic domain of human plasma kallikrein were modeled, as well as those with KLK4 and KLK7, and the structures were analyzed in order to identify the interactions that are responsible for inhibitory potency.

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.

A pilot study on the role of tissue kallikrein in the nafamostat-induced hyperkalemia.

A pilot study on the role of tissue kallikrein in the nafamostat-induced hyperkalemia.

Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii was recently been shown to be an effective bio-manufacturing platform for the production of recombinant proteins. The advantage of using C. reinhardtii is that it is fast to grow, inexpensive to culture, and relatively safe. However, the expression of foreign proteins is always low and difficult to purify in C. reinhardtii. Human kallikrein has the potential to be developed into certain drugs, like insulin. Therefore, its biosynthesis is important to drug development. In this study, we synthesized the sg gene, a signal peptide sequence of alkaline phosphatase, and inserted it into a pH124 plasmid, which contains a HSP70A-RBCS2 promoter and a RBCS2 terminator. Then, we inserted the human kallikrein gene klk1 behind the sg sequence to make a pHsgk124 vector. The pHsgk124 were transferred into a cell-wall deficient strain of C. reinhardtii, cc-503, by using the glass bead method. Southern blot analysis showed that sg and klk1 were incorporated into genes of the transgenic C. reinhardtii. RT-PCR analysis showed that it had an active transcription and its expression increased three times under heat stress. Western blot analyses of proteins inside and outside cells (in the culture medium) showed that klk1 was expressed in the cell and the resulting protein was secreted into medium. An enzyme activity assay showed that the recombinant protein had the ability to hydrolyze the specific substrate H-D-Val-Leu-Arg-Pna. In conclusion, we successfully bioengineered C. reinhardtii to produce and secrete human kallikrein protein, which has important biomedical implications.

EPITHELIAL CELL GENE NETWORKS UPREGULATED IN OBESE ASTHMATIC CHILDREN

Introduction Obesity is a major pediatric health concern affecting almost 20% of American children and adolescents. Obesity is a risk factor for the development of asthma and is associated with increased asthma severity. Children with obesity and asthma have reduced responsiveness to inhaled corticosteroids, increased use of systemic corticosteroids during exacerbations, and a reduced FEV1/FVC ratio. The mechanisms underlying the influence of obesity on asthma pathogenesis remain poorly understood. Methods Children (ages 6-17 years) with a priori defined exacerbation-prone asthma and blood eosinophils ≥150/mm3 had repeated nasal lavage sampling during a one-week period following reported URI symptoms. Nasal cell differentials were determined by cytospin and nasal gene expression assessed by RNA-sequencing. Differential gene expression was assessed by cell deconvolution and modular analysis. Results 154 URI events from 106 participants were captured and 47 of those events resulted in asthma exacerbation. 42/106 participants were obese defined by a BMI ≥95th percentile. Children with obesity had higher expression levels of 7 geneset modules when compared to those without obesity. These modules were associated with the airway epithelium and represent molecular functions including keratinization, tissue kallikrein induction, extracellular matrix production, and EGFR signaling. Measured effect sizes ranged from 1.24-2.05 fold higher. Conclusions We identified several airway epithelial associated gene networks upregulated in children with obesity and asthma. Previous studies of obesity and asthma have focused largely on metabolic and inflammatory alterations. Our results highlight the relevance of epithelial response pathways, which may serve as novel targets for biomarkers or therapies for obesity associated asthma.

Depsipeptides Featuring a Neutral P1 Are Potent Inhibitors of Kallikrein-Related Peptidase 6 with On-Target Cellular Activity.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins (KLKs). Many KLKs are investigated as potential biomarkers for cancer as well as therapeutic drug targets for a number of pathologies. KLK6, in particular, has been implicated in neurodegenerative diseases and cancer, but target validation has been hampered by a lack of selective inhibitors. This work introduces a class of depsipeptidic KLK6 inhibitors, discovered via high-throughput screening, which were found to function as substrate mimics that transiently acylate the catalytic serine of KLK6. Detailed structure-activity relationship studies, aided by in silico modeling, uncovered strict structural requirements for potency, stability, and acyl-enzyme complex half-life. An optimized scaffold, DKFZ-251, demonstrated good selectivity for KLK6 compared to other KLKs, and on-target activity in a cellular assay. Moreover, DKFZ-633, an inhibitor-derived activity-based probe, could be used to pull down active endogenous KLK6.

A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering

Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.

The Combination of Human Urinary Kallidinogenase and Mild Hypothermia Protects Adult Rats Against Hypoxic-Ischemic Encephalopathy-Induced Injury by Promoting Angiogenesis and Regeneration.

Objectives: Human Urinary Kallidinogenase (HUK) is a tissue kallikrein that plays neuroprotective role in ischemic conditions via different mechanisms. Mild hypothermia (MH) is another robust neuroprotectant that reduces mortality but does not profoundly ameliorate the neurological outcome in hypoxic-ischemic encephalopathy (HIE) patients. However, whether the combination of HUK and MH can be used as a promising neuroprotective treatment in HIE is unknown.Methods: One-hundred and forty-four adult Wistar rats were randomly divided into five groups: Sham, HIE, HUK, MH and a combination of HUK and MH treatment. The HIE rat model was established by right carotid dissection followed by hypoxia aspiration. The survival curve was created within 7 days, and the neurological severity scores (NSS) were assessed at days 0, 1, 3, and 7. Nissl staining, Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), immunofluorescent staining and western blotting were used to evaluate neuronal survival, apoptosis and necrosis, tight-junction proteins Claudin-1 and Zonula occludens-1 (ZO-1), vascular endothelial growth factor (VEGF), doublecortex (DCX), bradykinin receptor B1 (BDKRB1), BDKRB2 and Ki67 staining.Results: The combined treatment rescued all HIE rats from death and had a best survival curve compared to HIE. The Combination also reduced the NSS scores after HIE at days 7, better than HUK or MH alone. The combination of HUK and MH reserved more cells in Nissl staining and inhibited neuronal apoptosis and necrosis as well as significantly attenuated HIE-induced decreases in claudin-1, ZO-1, cyclin D1 and BDKRB1/B2 in comparison to HUK or MH treatment alone. Moreover, the combined treatment increased the expression of VEGF and DCX as well as the number of Ki67-labeled cells.Conclusions: This study demonstrates that both HUK and MH are neuroprotective after HIE insult; however, the combined therapy with HUK and MH enhanced the efficiency and efficacy of either therapy alone in the treatment of HIE, at least partially by promoting angiogenesis and regeneration and rescuing tight-junction loss. The combination of HUK and MH seems to be a feasible and promising clinical strategy to alleviate cerebral injury following HIE insult.Highlights: -The combination of HUK and MH distinctly reduces neurological dysfunction in HIE rats.-HUK enhances the neuroprotective effects of MH in HIE.-MH attenuates tight-junction disruption, upregulates the BDKR B1/2, DCX and cyclin D1.-The combination of MH and HUK enhances the expressions of MH/HUK mediated-BDKR B1/2, DCX, cyclin D1 and Ki67 positive cells.

325 Preserved erectile function in the hyperhomocysteinaemia transgenic rats harboring human tissue kallikrein 1

To investigate the detailed mechanism of erectile dysfunction (ED) induced by hyperhomocysteinaemia (HHcy) in rats and determine whether the Human Tissue Kallikrein 1 (hKLK1) might improve it, as we have proved the protective role of hKLK1 on erectile function in aged rats. We established a rat model of HHCy through dietary-rich methionine (Met) in male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 10 weeks of age. Then 24 WTRs were divided into control (n=8), the low-dose (4% Met, n=8), and the high-dose (7% Met, n=8). Another 8 age-matched TGRs with the high-dose formed the TGR+7%Met group. 30 days later, erectile function, level of total homocysteinaemia (tHcy), oxidative stress, endothelial function, cavernous nerve function and fibrosis of all groups were determined. hKLK1 in the TGR+7%Met group could greatly decrease the tHcy levels and improve ED induced by HHcy in rats. For the endothelial function, hKLK1 could preserve the endothelial cell-cell junction, enhance endothelial regeneration and activated the Akt/eNOS signaling pathway. Together with the promotion of hKLK1 on nNOS expression, the NO/cGMP signaling pathway activity was also increased. For the fibrosis, hKLK1 could preserve normal corpus cavernosum structure through inhibiting apoptosis and promoting autophagy on corpus cavernosum smooth muscle cells. In addition, hKLK1 also inhibited the fibrosis-related signaling pathway activity.

Expression profile of human tissue kallikrein 15 provides preliminary insights into its roles in the prostate and testis

BackgroundHuman tissue kallikrein 15 (KLK15) is the latest member of the kallikrein-related peptidase family. Little is known about the pathophysiological roles of KLK15. Previous studies implied a role of KLK15 in prostate cancer. MethodsIn the present study, we examined KLK15 protein expression using a new immunoassay (ELISA) and immunohistochemistry (IHC). ResultsHighest KLK15 levels were detected in the testis and seminal fluid, whereas lower levels were observed in prostate and other tissues. Immunohistochemical analysis of testis suggests that KLK15 is strongly expressed in mature spermatids, but not in immature germ cells. KLK15 displayed predominantly nuclear localization in the basal cell layer of the prostatic epithelium. We also measured KLK15 in supernatants of various cell lines. Highest KLK15 levels were primarily detected in prostate cancer cell lines and KLK15 expression was hormone-independent, in contrast to KLK3. ConclusionsCollectively, our data provide insights into the localization and possible role of KLK15 in human physiology.

Human tissue kallikrein 1 ameliorates erectile function via modulation of macroautophagy in aged transgenic rats.

Previously, we have demonstrated that human tissue kallikrein 1 (hKLK1) improves age-related erectile dysfunction (ED). Autophagy has been implicated in age-related diseases, including ED. However, the molecular mechanisms underlying hKLK1-mediated amelioration of age-related ED via regulation of autophagy remains unknown. To explore the potential mechanism, male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring human KLK1 (TGR) were bred till 4 or 18months of age and divided into three groups: young WTR (yWTR) as the control group, aged WTR (aWTR) group, and aged TGR (aTGR) group. The erectile function of each rat was evaluated using cavernous nerve electrostimulation. The ratio of intracavernous pressure/mean arterial pressure (ICP/MAP) and total ICP were also measured. Western blotting, immunohistochemistry, and transmission electron microscopy were performed to detect the levels of autophagy. The expression levels of related signaling pathways were determined by western blotting and immunohistochemistry. We found that hKLK1 improved the impaired erectile function of aged rats. Compared to the yWTR and aTGR groups, the aWTR group showed reduced smooth muscle/collagen ratio, fewer autophagosomes, and lower expression of Beclin 1 and LC3-II, which indicate impaired smooth muscle function and low level of autophagy in the smooth muscle cells. Moreover, the PI3K/Akt/mTOR signaling pathway, which is considered to be a negative regulator of autophagy, was upregulated in the aWTR group. hKLK1 may partially restore erectile function in aged transgenic rats by upregulating protective autophagy via the PI3K/Akt/mTOR pathway. These observations indicate that hKLK1 is a potential gene therapy candidate for age-related ED.

Biochemical characterization of human tissue kallikrein 15 and examination of its potential role in cancer

ObjectiveHuman tissue kallikrein 15 (KLK15) is the last cloned member of the KLK-related gene family. Despite being implicated in multiple cancers, its pathophysiological role remains unknown. We aimed to biochemically characterize KLK15 and preliminarily study its role in cancer. Design & methodsRecombinant KLK15 protein was produced, purified to homogeneity and quantified by mass spectrometry (parallel reaction monitoring analysis). We profiled the enzymatic activity of KLK15 using fluorogenic peptide substrates, and performed kinetic analysis to discover the cleavage sites. As KLK15 has mainly been associated with prostate cancer, we used a degradomic approach and subsequent KEGG pathway analysis to identify a number of putative protein substrates in the KLK15-treated prostate cancer cell line PC3. ResultsWe discovered trypsin-like activity in KLK15, finding that it cleaves preferentially after arginine (R). The enzymatic activity of KLK15 was regulated by different factors such as pH, cations and serine protease inhibitors. Notably, we revealed that KLK15 most likely interacts with the extracellular matrix (ECM) receptor group. ConclusionTo our knowledge, this is the first study that experimentally verifies the trypsin-like activity of KLK15. We show here for the first time that KLK15 may be able to cleave many ECM components, similar to several members of the KLK family. Thus the protease could potentially be linked to tumorigenesis by promoting metastasis via this mechanism.

Overview of tissue kallikrein and kallikrein-related peptidases in breast cancer.

The kallikrein family comprises tissue kallikrein and 14 kallikrein-related peptidases (KLKs) recognized as a subgroup of secreted trypsin- or chymotrypsin-like serine proteases. KLKs are expressed in many cellular types where they regulate important physiological activities such as semen liquefaction, immune response, neural development, blood pressure, skin desquamation and tooth enamel formation. Tissue kallikrein, the oldest member and kinin-releasing enzyme, and KLK3/PSA, a tumor biomarker for prostate cancer are the most prominent components of the family. Additionally, other KLKs have shown an abnormal expression in neoplasia, particularly in breast cancer. Thus, increased levels of some KLKs may increase extracellular matrix degradation, invasion and metastasis; other KLKs modulate cell growth, survival and angiogenesis. On the contrary, KLKs can also inhibit angiogenesis and produce tumor suppression. However, there is a lack of knowledge on how KLKs are regulated in tumor microenvironment by molecules present at the site, namely cytokines, inflammatory mediators and growth factors. Little is known about the signaling pathways that control expression/secretion of KLKs in breast cancer, and further how activation of PAR receptors may contribute to functional activity in neoplasia. A better understanding of these molecular events will allow us to consider KLKs as relevant therapeutic targets for breast cancer.

Therapeutic Values of Human Urinary Kallidinogenase on Cerebrovascular Diseases.

The term “tissue kallikrein” is used to describe a group of serine proteases shared considerable sequence homology and colocalize in the same chromosomal locus 19q13. 2–q13.4. It has been widely discovered in various tissues and has been proved to be involved in kinds of pathophysiological processes, such as inhibiting oxidative stress, inflammation, apoptosis, fibrosis and promoting angiogenesis, and neurogenesis. Human Urinary Kallidinogenase (HUK) extracted from human urine is a member of tissue kallikrein which could convert kininogen to kinin and hence improve the plasma kinin level. Medical value of HUK has been widely investigated in China, especially on acute ischemic stroke. In this review, we will summarize the therapeutic values of Human Urinary Kallidinogenase on acute ischemic stroke and its potential mechanisms.

Molecular mechanism of the role of carbamyl erythropoietin in treating diabetic retinopathy rats.

The aim of the present study was to investigate the therapeutic effects of carbamyl erythropoietin (CEPO) and safflor yellow (SY) in the treatment of rats with diabetic retinopathy (DR) as well as exploring the mechanism of action. Male SD rats were used to establish a diabetes model and streptozotocin-induced retinopathy was also performed in rats. A total of 126 rats with DR were obtained, and model rats were randomly divided into the model (n=42), experimental (n=42) and control (n=42) groups. The rats in the model group were injected with saline, the rats in the experimental group were treated with CEPO, and the rats in the control group were treated with SY. After treatment for 2 weeks, the retinas were harvested for quantitative analysis of the mRNA expression levels of angiogenesis-promoting and -inhibiting molecules, apoptosis-promoting and -inhibiting molecules, and oxidative stress pathway-related factors by Reverse transcription-quantitative PCR (RT-qPCR). No significant differences in expression levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), angiopoietin (Ang-1), tissue kallikrein (TKLK) and pigment epithelium-derived factor (PEDF) were observed between the experimental and model groups (P>0.05). The expression levels of apoptosis-promoting molecules Bcl-2 related X protein (Bax) and cysteine aspartate specific protease (caspase-3) mRNA in the retina of the experimental group was significantly lower than those in the control group (P<0.05). The expression levels of Bcl-2 and survivin mRNA were significantly higher in the experimental group than in the control group (P<0.05). The expression levels of the oxidative stress pathway nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) mRNA were significantly higher in the experimental group than in the control group. Therefore, the therapeutic effects of CEPO in treating DR are better than those of SY. As a result, CEPO may inhibit apoptosis and oxidative stress damage of retinal tissue cells in DR rats without affecting angiogenesis.

Discovery of novel transcripts of the human tissue kallikrein (KLK1) and kallikrein-related peptidase 2 (KLK2) in human cancer cells, exploiting Next-Generation Sequencing technology

Tissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional properties. Several KLK transcripts have been found aberrantly expressed in numerous human malignancies, confirming their prognostic or/and diagnostic values. However, the process of alternative splicing can now be studied in-depth due to the development of Next-Generation Sequencing (NGS). In the present study, we used NGS to discover novel transcripts of the KLK1 and KLK2 genes, after nested touchdown PCR. Bioinformatics analysis and PCR experiments revealed a total of eleven novel KLK transcripts (two KLK1 and nine KLK2 transcripts). In addition, the expression profiles of each novel transcript were investigated with nested PCR experiments using variant-specific primers. Since KLKs are implicated in human malignancies, qualifying as potential biomarkers, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer.

Kallikrein-related peptidases in lung diseases.

Human tissue kallikreins (KLKs) are 15 members of the serine protease family and are present in various healthy human tissues including airway tissues. Multiple studies have revealed their crucial role in the pathophysiology of a number of chronic, infectious and tumour lung diseases. KLK1, 3 and 14 are involved in asthma pathogenesis, and KLK1 could be also associated with the exacerbation of this inflammatory disease caused by rhinovirus. KLK5 was demonstrated as an influenza virus activating protease in humans, and KLK1 and 12 could also be involved in the activation and spread of these viruses. KLKs are associated with lung cancer, with up- or downregulation of expression depending on the KLK, cancer subtype, stage of tumour and also the microenvironment. Functional studies showed that KLK12 is a potent pro-angiogenic factor. Moreover, KLK6 promotes malignant-cell proliferation and KLK13 invasiveness. In contrast, KLK8 and KLK10 reduce proliferation and invasion of malignant cells. Considering the involvement of KLKs in various physiological and pathological processes, KLKs appear to be potential biomarkers and therapeutic targets for lung diseases.

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

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. Left middle cerebral artery occlusion (MCAO) was performed to generate the rat model of ischemic stroke, followed by daily RIPostC treatment for maximum 21days. 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. Daily RIPostC for more than 21days 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. 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.

Kallikrein–Kinin System Suppresses Type I Interferon Responses: A Novel Pathway of Interferon Regulation

The Kallikrein–Kinin System (KKS), comprised of kallikreins (klks), bradykinins (BKs) angiotensin-converting enzyme (ACE), and many other molecules, regulates a number of physiological processes, including inflammation, coagulation, angiogenesis, and control of blood pressure. In this report, we show that KKS regulates Type I IFN responses, thought to be important in lupus pathogenesis. We used CpG (TLR9 ligand), R848 (TLR7 ligand), or recombinant IFN-α to induce interferon-stimulated genes (ISGs) and proteins, and observed that this response was markedly diminished by BKs, klk1 (tissue kallikrein), or captopril (an ACE inhibitor). BKs significantly decreased the ISGs induced by TLRs in vitro and in vivo (in normal and lupus-prone mice), and in human PBMCs, especially the induction of Irf7 gene (p < 0.05), the master regulator of Type I IFNs. ISGs induced by IFN-α were also suppressed by the KKS. MHC Class I upregulation, a classic response to Type I IFNs, was reduced by BKs in murine dendritic cells (DCs). BKs decreased phosphorylation of STAT2 molecules that mediate IFN signaling. Among the secreted pro-inflammatory cytokines/chemokines analyzed (IL-6, IL12p70, and CXCL10), the strongest suppressive effect was on CXCL10, a highly Type I IFN-dependent cytokine, upon CpG stimulation, both in normal and lupus-prone DCs. klks that break down into BKs, also suppressed CpG-induced ISGs in murine DCs. Captopril, a drug that inhibits ACE and increases BK, suppressed ISGs, both in mouse DCs and human PBMCs. The effects of BK were reversed with indomethacin (compound that inhibits production of PGE2), suggesting that BK suppression of IFN responses may be mediated via prostaglandins. These results highlight a novel regulatory mechanism in which members of the KKS control the Type I IFN response and suggest a role for modulators of IFNs in the pathogenesis of lupus and interferonopathies.