Kallistatin Research Articles

Kallistatin and Glypican-3 as Reliable Biomarkers for the Prediction of Liver Cirrhosis

Background: Cirrhosis is a condition in which the liver does not function properly due to long-term damage, this damage is characterized by the replacement of normal liver tissue by scar tissue. Objective: In the present study, an attempt was made to explore the role of kallistatin (KAL) and glypican3 (GPC3) as a predictor of liver cirrhosis (LC) in patients with hepatitis, non-alcoholic fatty acid and autoimmune diseases. Materials and Methods: This case–control study included 98 cirrhotic patients (34 patients with hepatitis B & A, 31 patients with non-alcoholic fatty liver disease and 33 patients with autoimmune) and 30 apparently healthy individuals. The laboratory investigations were performed, and the serum KAL and GPC3 were measured in all volunteers. Results: Serum levels of KAL, GPC3, and TAC were significantly decreased and MDA increased (p<0.01) in patients with LC as compared to control. The significant difference can also be indicatly seen in patients with hepatitis > non-alcoholic fatty acid > autoimmune diseases, respectively. The area under the curve (AUC) results obtained indicate that Kal and GPC3 could potentially be used as greater predictive biomarkers in LC with hepatitis ˃ NAFLD ˃ autoimmune diseases (Kal: AUC= -0.97, -0.91, -0.88; Gyp3: AUC= 0.89, 0.84, 0.79, respectively). Conclusion: Incorporating KAL and GPC3 screening into routine check-ups for patients with hepatitis, non-alcoholic fatty liver, and autoimmune diseases could aid in the early detection and prevention of LC-associated complications.

Elevated Kallistatin promotes the occurrence and progression of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, and the development of non-alcoholic steatohepatitis (NASH) might cause irreversible hepatic damage. Hyperlipidemia (HLP) is the leading risk factor for NAFLD. This study aims to illuminate the causative contributor and potential mechanism of Kallistatin (KAL) mediating HLP to NAFLD. 221 healthy control and 253 HLP subjects, 62 healthy control and 44 NAFLD subjects were enrolled. The plasma KAL was significantly elevated in HLP subjects, especially in hypertriglyceridemia (HTG) subjects, and positively correlated with liver injury. Further, KAL levels of NAFLD patients were significantly up-regulated. KAL transgenic mice induced hepatic steatosis, inflammation, and fibrosis with time and accelerated inflammation development in high-fat diet (HFD) mice. In contrast, KAL knockout ameliorated steatosis and inflammation in high-fructose diet (HFruD) and methionine and choline-deficient (MCD) diet-induced NAFLD rats. Mechanistically, KAL induced hepatic steatosis and NASH by down-regulating adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) by LRP6/Gɑs/PKA/GSK3β pathway through down-regulating peroxisome proliferator-activated receptor γ (PPARγ) and up-regulating kruppel-like factor four (KLF4), respectively. CGI-58 is bound to NF-κB p65 in the cytoplasm, and diminishing CGI-58 facilitated p65 nuclear translocation and TNFα induction. Meanwhile, hepatic CGI-58-overexpress reverses NASH in KAL transgenic mice. Further, free fatty acids up-regulated KAL against thyroid hormone in hepatocytes. Moreover, Fenofibrate, one triglyceride-lowering drug, could reverse hepatic steatosis by down-regulating KAL. These results demonstrate that elevated KAL plays a crucial role in the development of HLP to NAFLD and may be served as a potential preventive and therapeutic target.

Kallistatin Deficiency Induces the Oxidative Stress-Related Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells: A Novel Protagonist in Age-Related Macular Degeneration.

Retinal pigment epithelium (RPE) dysfunction induced by oxidative stress-related epithelial-mesenchymal transition (EMT) of RPE is the primary underlying mechanism of age-related macular degeneration (AMD). Kallistatin (KAL) is a secreted protein with an antioxidative stress effect. However, the relationship between KAL and EMT in RPE has not been determined. Therefore we aimed to explore the impact and mechanism of KAL in oxidative stress-induced EMT of RPE. Sodium iodate (SI) was injected intraperitoneally to construct the AMD rat model and investigate the changes in RPE morphology and KAL expression. KAL knockout rats and KAL transgenic mice were used to explain the effects of KAL on EMT and oxidative stress. In addition, Snail overexpressed adenovirus and si-RNA transfected ARPE19 cells to verify the involvement of Snail in mediating KAL-suppressed EMT of RPE. AMD rats induced by SI expressed less KAL in the retina, and KAL knockout rats showed RPE dysfunction spontaneously where EMT and reactive oxygen species (ROS) production increased in RPE. In contrast, KAL overexpression attenuated EMT and ROS levels in RPE, even in TGF-β treatment. Mechanistically, Snail reversed the beneficial effect of KAL on EMT and ROS reduction. Moreover, KAL ameliorated SI-induced AMD-like pathological changes. Our findings demonstrated that KAL inhibits oxidative stress-induced EMT by downregulating the transcription factor Snail. Herein, KAL knockout rats may be an appropriate animal model for observing spontaneous RPE dysfunction for AMD-like retinopathy, and KAL may represent a novel therapeutic target for treating dry AMD.

Kallistatin as an inhibitory protein against colorectal cancer cells through binding to LRP6

Kallistatin (KL) is a member of the serine proteinase inhibitor (serpin) family regulating oxidative stress, vascular relaxation, inflammation, angiogenesis, cell proliferation, and invasion. The heparin-binding site of Kallistatin has an important role in the interaction with LRP6 leading to the blockade of the Wnt signaling pathway. In this study, we aimed to explore the structural basis of the Kallistatin-LRP6E1E4 complex using in silico approaches and evaluating the anti-proliferative, apoptotic, and cell cycle arrest activities of Kallistatin in colon cancer lines. The molecular docking showed Kallistatin could bind to the LRP6E3E4 much stronger than LRP6E1E2. The Kallistatin-LRP6E1E2 and Kallistatin-LRP6E3E4 complexes were stable during Molecular Dynamics (MD) simulation. The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) showed that the Kallistatin-LRP6E3E4 has a higher binding affinity compared to Kallistatin-LRP6E1E2. Kallistatin induced higher cytotoxicity and apoptosis in HCT116 compared to the SW480 cell line. This protein-induced cell-cycle arrest in both cell lines at the G1 phase. The B-catenin, cyclin D1, and c-Myc expression levels were decreased in response to treatment with Kallistatin in both cell lines while the LRP6 expression level was decreased in the HCT116 cell line. Kallistatin has a greater effect on the HCT116 cell line compared to the SW480 cell line. Kallistatin can be used as a cytotoxic and apoptotic-inducing agent in colorectal cancer cell lines.

Kallistatin prevents ovarian hyperstimulation syndrome by regulating vascular leakage.

Angiogenesis and increased permeability are essential pathological basis for the development of ovarian hyperstimulation syndrome (OHSS). Kallistatin (KS) is an endogenous anti‐inflammatory and anti‐angiogenic factor that participates in a variety of diseases, but its role in OHSS remains unknown. In this study, treating a human ovarian granulosa‐like tumour cell line KGN and human primary granulosa cells (PGCs) with human chorionic gonadotropin (hCG) reduced the expression of KS, but increased the expression of VEGF. Furthermore, we found that KS could attenuate the protein level of VEGF in both KGN cells and human PGCs. More interestingly, we observed that exogenous supplementation of KS significantly inhibited a series of signs of OHSS in mice, including weight gain, ovarian enlargement, increased vascular permeability and up‐regulation of VEGF expression. In addition, KS was proved to be safe on mice ovulation, progression of normal pregnancy and fetus development. Collectively, these findings demonstrated that KS treatment prevented OHSS, at least partially, through down‐regulating VEGF expression. For the first time, these results highlight the potential preventive value of KS in OHSS.

Kallistatin in follicular fluid of women with endometriosis and its correlation with IVF outcome

Background Endometriosis (EM) affects 10% women of reproductive age and alters fertility. Its management is still debated notably the timing of surgery and ART in infertility. Kallistatin (KS) is an endogenous protein that regulates differential signaling pathways and biological functions. However, the function and the underlying molecular mechanism in EM and its correlation with in vitro fertilization (IVF) outcome have not been determined. The purpose of this study was to evaluate KS concentrations in follicular fluid (FF) of women with EM and controls women without EM who underwent IVF with embryo transfer (IVF–ET). Methods FF KS concentrations from 40 patients with EM and 40 non-EM patients were measured by ELISA. Results Compared with the non-EM patients, patients with EM had lower KS levels in FF (281.67 ± 104.60 vs. 490.70 ± 216.33 pg/ml). The rates of fertilization (61.64 ± 22.42 vs. 71.00 ± 24.39%), available embryo (45.96 ± 19.83 vs. 50.61 ± 26.26%), and top-quality embryo (12.71 ± 21.01 vs. 16.04 ± 16.87%) were significantly lower in the EM group than in the control group. The KS concentrations in the FF of women who conceived consequent to the treatment were significantly higher than those from women who did not in the combined EM and control groups. Conclusions These results indicate that the KS concentration in FF could be used as a predictor for IVF–ET outcomes. This may contribute to the pathologic mechanism responsible for the poor outcome of IVF in patients with EM.

Kallistatin Inhibits Anoikis Resistance and Metastasis of Ectopic Endometrium Cells by Modulating MnSOD and Caspase 3 Signaling.

Endometriosis (EM) is a disease that involves active endometrial cell invasion and migration which is an important reason for infertility. Anoikis resistance is the most important prerequisite for EM, but the molecular mechanism is not yet clear. Kallistatin (KS) is one kind of serine protease inhibitors which had extensive biological function including anti-inflammatory, antioxidant stress, anti-angiogenesis, and anti-tumor. Our preliminary data showed that the level of KS in EM patients' endometrial tissue and blood were much lower than control (non-EM) patients without endometriosis. Interestingly, the decrease of KS is correlated with the severity of endometriosis. Moreover, kallistatin recombinant protein could increase the anoikis rate of ectopic endometrium cells (EESCs), and then inhibits its metastasis and invasion. Mechanically, our data show that the EESCs have lower intracellular reactive oxygen species (ROS) production and KS can elevate the ROS levels significantly. Further, KS modulate expression of MnSOD and caspase 3 signaling in EESCs grown in suspended conditions. These findings reveal novel mechanisms of KS in inducing anoikis and metastasis in EESCs, thus inhibiting EM progression by regulation of MnSOD and caspase 3 signaling. Our findings suggest that KS is a significant protein with prospects for application in EM.

Kallistatin alleviates heart failure in rats by inhibiting myocardial inflammation and apoptosis via regulating sirt1.

Heart failure (HF) is the loss of myocardial structure and function caused by various congenital or acquired heart diseases. This study explored the new target of treatment of HF by investigating the effect of Kallistatin (KS) on inflammation and apoptosis of myocardial tissue in HF rats. We used doxorubicin to induce rat HF, and determined the success rate of modeling by detecting changes in rat heart weight and body weight, cardiac function and histology. We used two different doses (1 mg/kg, 2 mg/kg) of KS intraperitoneally injected rats and detected changes in inflammation and apoptosis of rat myocardial tissue by enzyme-linked immunosorbent assay (ELISA), Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemical staining. Changes in the expression of sirt1 were also detected. In addition, we cultured rat myocardial cell line, H9c2 cells, and used siRNA-sirt1 to inhibit sirt1 in H9c2 cells to clarify the mechanism of KS regulating myocardial cells. The body weight of HF rats treated with KS decreased while the heart weight increased. KS has also been found to reduce the concentration of brain natriuretic polypeptide (BNP) in rat serum. The results of echocardiography showed that KS effectively relieved the cardiac function of HF rats. Inflammatory factors (interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α) and pro-apoptotic molecules (caspase3/9 and Bax) in the serum and myocardial tissue of rats treated with KS were also significantly reduced. The inhibition of sirt1 in H9c2 cells significantly reduced the anti-apoptotic effect of KS on H9c2 cells. KS reduces the inflammation and apoptosis of myocardial tissue in HF rats by promoting the expression of sirt1, thereby alleviating HF-induced myocardial injury.

Kallistatin correlates with inflammation in abdominal aortic aneurysm and suppresses its formation in mice.

Kallistatin (KS), encoded by SERPINA4, was suggested to play a protective role in many cardiovascular diseases. However, its role in the pathogenesis of abdominal aortic aneurysm (AAA) remains unclear. The aim of this study was to examine the potential association of KS with AAA pathogenesis. We examined KS (SERPINA4) expression in human AAA by PCR, immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA) and analyzed correlations between kallistain and clinical data. We then analyzed the effect of recombinant KS on AAA formation and the Wingless (Wnt) signaling pathway in a mouse AAA model developed by angiotensin II (AngII) infusion to apolipoprotein E-deficient (ApoE-/-) mice. In AAA tissue samples, KS was significantly increased compared with samples from the control group (P<0.001, P<0.001, respectively). Clinically, decreased SERPINA4 expression in AAA tissue samples represented an increased rate of iliac artery aneurysm [odds ratio (OR): 0.017; P=0.040]. And decreased plasma KS level represented a high risk for rupture (OR: 0.837; P=0.034). KS inhibited AAA formation and blocked the Wnt signaling pathway in AngII-infused ApoE-/- mice. The present study demonstrates that aberrant changes in KS expression occur in AAA. KS plays an important anti-inflammatory role and showed important clinical correlations in AAA. Decreased KS (SERPINA4) level is a risk factor of AAA rupture. Our pre-clinical animal experiments indicate that treatment with recombination KS suppresses AngII-induced aortic aneurysm formation and might be a new target for the drug therapy of AAA.

Kallistatin inhibits tumour progression and platinum resistance in high-grade serous ovarian cancer

Ovarian cancer is the most lethal gynaecologic malignancy. Although there are various subtypes of ovarian cancer, high-grade serous ovarian cancer (HGSOC) accounts for 70% of ovarian cancer deaths. Chemoresistance is the primary reason for the unfavourable prognosis of HGSOC. Kallistatin (KAL), also known as SERPINA4, is part of the serpin family. Kallistatin has been discovered to exert multiple effects on angiogenesis, inflammation and tumour progression. However, the roles and clinical significance of kallistatin in HGSOC remain unclear. Here, we showed that kallistatin was significantly downregulated in HGSOC compared to normal fallopian tube (FT) tissues. Low expression of kallistatin was associated with unfavourable prognosis and platinum resistance in HGSOC. Overexpression of kallistatin significantly inhibited proliferation and metastasis, and enhanced platinum sensitivity and apoptosis in ovarian cancer cells. Collectively, these findings demonstrate that kallistatin serves as a prognostic predictor and provide a potential therapeutic target for HGSOC.

Kallistatin Inhibits Atherosclerotic Inflammation by Regulating Macrophage Polarization.

Kallistatin (KS) has been recognized as a plasma protein with anti-inflammatory functions. Macrophages are the primary inflammatory cells in atherosclerotic plaques. However, it is unknown whether KS plays a role in macrophage development and the pathogenesis of atherosclerosis. This study investigated the role of KS in macrophage development, a key pathological process in atherosclerosis. An atherosclerosis model was established in ApoE-/- mice via partial left carotid artery (PLCA) ligation. An adenovirus vector (Ad. HKS) containing the human KS gene was delivered via the tail vein before PLCA ligation. The mice were divided into two groups: the PLCA + Ad. HKS and PLCA + adenovirus vector (Ad. Null) groups and followed for 2 and 4 weeks. Human KS was expressed in the mice after KS gene delivery. In addition, KS significantly inhibited plaque formation and reduced inflammation in the plaques and liver 4 weeks after gene delivery. Moreover, KS gene delivery significantly increased the expression of interleukin-10 and Arginase 1, which are M2 macrophage markers, and reduced the expression of inducible nitric oxide synthase and monocyte chemotactic protein 1, which are M1 macrophage markers. Furthermore, in cultured RAW 264.7 macrophages, KS significantly stimulated M2 marker expression and differentiation and decreased M1 marker expression, as determined by flow cytometry and real-time polymerase chain reaction. These effects were blocked by Krüppel-like factor 4 small-interfering RNA oligonucleotides. These findings demonstrate that KS inhibits atherosclerotic plaque formation and regulates M1/M2 macrophage polarization via Krüppel-like factor 4 activation.

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 …

Plasma Kallistatin is Associated with Leukocyte Telomere Length in Young African Americans

ObjectiveLarge population studies demonstrate that African Americans versus their Caucasian counterparts have a steeper decline in leukocyte telomere lengths (LTL) with age. Telomere shortening plays an important role in cellular aging. Kallistatin, a novel tissue kallikrein inhibitor, has anti‐inflammatory and anti‐oxidant properties. Recently we have shown that kallistatin inhibited vascular injury and cell apoptosis in animal models and cultured cells. Thus we aimed to test our hypothesis that plasma kallistatin levels are positively associated with LTL in African Americans.MethodsLeukocyte telomere lengths (the relative ratio of telomere repeat copy number (T) to single‐copy gene copy number (S)) were determined by a quantitative polymerase chain reaction method in 188 (60 males and 128 females) apparently healthy young African American subjects aged 13 to 49 years. Plasma kallistatin levels were measured by enzyme‐linked immunosorbent assay.ResultsCompared to males, females were older (24.0 ± 9.19 vs 17.9 ± 4.5 years, p&lt;0.001), and had slightly shorter LTL (T/S ratio 1.00 ± 0.23 vs 1.09 ± 0.27, p= 0.03). In the whole sample, plasma kallistatin levels are positively and significantly associated with LTL (r=0.31, p&lt;0.001). The significance persisted after adjusting for age, sex, and body mass index (r= 0.16, p=0.04).ConclusionThis is the first study to examine the relationship between kallistatin and telomeres in humans. Kallistatin may have anti‐cellular aging effect, which may be attributed to its anti‐inflammatory and anti‐oxidant properties. These finding indicate that plasma kallistatin might serve as a biomarker for cellular aging in humans.Support or Funding InformationHZ and YD are supported by NHLBI PO1 HL69999 and AHA 14GRANT20480211.

Effects of kallistatin on oxidative stress and inflammation on renal ischemia-reperfusion injury in mice.

Kallistatin (KS) is a serine proteinase. The result of KS on 'Renal Ischemia- Reperfusion Injury' (IRI) has not clearly been researched. In this study, investigative research has been conducted to draw results on the administration of human KS on kidney response conducted within a mouse model of IRI. BALB/c mice were used and given 30 min ischemia that was injected into the kidney which was followed with 24 h reperfusion. The human KS gene contained within an adenoviral vector was injected intravenously 30 min before reperfusion and 12h after reperfusion. Analyses illustrated what impact KS had on renal IRI in realtion to tubular necrosis, apoptotic cell death, inflammatory cytokines, renal function, and inflammatory cell infiltration. KS gene transfer significantly had a positive impact on renal function (reduced blood urea-nitrogen: 73.5±13.6 vs. 195.4±14.6 mg/dL at day three, p < 0.05 and the serum creatinine levels: 0.23±0.02 vs. 0.71±0.14 mg/dL at day three, p < 0.05), reduced tubular necrosis and apoptosis of IRI kidneys. The permeation of cells that were inflamed and the manufacturing of pro-inflammatory cytokines (RANTES-is regulated through been activated with normal T-cell which are expressed and secreted, tumour necrosis interleukin-1β factor-α, and interferon-γ) resulted in significantly suppressing KS in mice with IRI. The efficacy to scavenge superoxide in tubule cells was also demonstrated by high-performance liquid chromatography. Our study suggests a novel role of KS in renal protection after 'Renal Ischemia-Reperfusion Injury' blocking of oxidative stress and renal inflammation.

Kallistatin Modulates Immune Cells and Confers Anti-Inflammatory Response To Protect Mice from Group A Streptococcal Infection

Group A streptococcus (GAS) infection may cause severe life-threatening diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Despite the availability of effective antimicrobial agents, there has been a worldwide increase in the incidence of invasive GAS infection. Kallistatin (KS), originally found to be a tissue kallikrein-binding protein, has recently been shown to possess anti-inflammatory properties. However, its efficacy in microbial infection has not been explored. In this study, we transiently expressed the human KS gene by hydrodynamic injection and investigated its anti-inflammatory and protective effects in mice via air pouch inoculation of GAS. The results showed that KS significantly increased the survival rate of GAS-infected mice. KS treatment reduced local skin damage and bacterial counts compared with those in mice infected with GAS and treated with a control plasmid or saline. While there was a decrease in immune cell infiltration of the local infection site, cell viability and antimicrobial factors such as reactive oxygen species actually increased after KS treatment. The efficiency of intracellular bacterial killing in neutrophils was directly enhanced by KS administration. Several inflammatory cytokines, including tumor necrosis factor alpha, interleukin 1β, and interleukin 6, in local infection sites were reduced by KS. In addition, KS treatment reduced vessel leakage, bacteremia, and liver damage after local infection. Therefore, our study demonstrates that KS provides protection in GAS-infected mice by enhancing bacterial clearance, as well as reducing inflammatory responses and organ damage.

Depletion of endogenous kallistatin exacerbates renal and cardiovascular oxidative stress, inflammation, and organ remodeling

Kallistatin (KS) levels are reduced in the kidney and blood vessels under oxidative stress conditions. To determine the function of endogenous KS in the renal and cardiovascular systems, KS levels were depleted by daily injection of anti-rat KS antibody into DOCA-salt hypertensive rats for 10 days. Administration of anti-KS antibody resulted in reduced KS levels in the circulation but increased levels of serum thiobarbituric acid reactive substances (an indicator of lipid peroxidation) as well as superoxide formation in the aorta. Moreover, anti-KS antibody injection resulted in increased NADH oxidase activity and superoxide production but decreased nitric oxide levels in the kidney and heart. Endogenous KS blockade aggravated renal dysfunction, damage, hypertrophy, inflammation, and fibrosis as evidenced by decreased creatinine clearance and increased serum creatinine, blood urea nitrogen and urinary protein levels, tubular dilation, protein cast formation, glomerulosclerosis, glomerular enlargement, inflammatory cell accumulation, and collagen deposition. In addition, rats receiving anti-KS antibody had enhanced cardiac injury as indicated by cardiomyocyte hypertrophy, inflammation, myofibroblast accumulation, and fibrosis. Renal and cardiac injury caused by endogenous KS depletion was accompanied by increases in the expression of the proinflammatory genes tumor necrosis factor-α and intercellular adhesion molecule-1 and the profibrotic genes collagen I and III, transforming growth factor-β, and tissue inhibitor of metalloproteinase-1. Taken together, these results implicate an important role for endogenous KS in protection against salt-induced renal and cardiovascular injury in rats by suppressing oxidative stress, inflammation, hypertrophy, and fibrosis.

Inhibition of angiogenesis and HCT-116 xenograft tumor growth in mice by kallistatin

To investigate the inhibitory effect of kallistatin (KAL) on angiogenesis and HCT-116 xenograft tumor growth. Heterotopic tumors were induced by subcutaneous injection of 2 multiply 10(6) HCT-11 cells in mice. Seven days later, 2 multiply 10(11) rAAV-GFP or rAAV-KAL was injected intratumorally (n = 5 for each group). The mice were sacrificed at d 28, by which time the tumors in the rAAV-GFP group had grown to beyond 5% of the total body weight. Tumor growth was measured by calipers in two dimensions. Tumor angiogenesis was determined with tumor microvessel density (MVD) by immunohistology. Tumor cell proliferation was assessed by Ki-67 staining. Intratumor injection of rAAV-KAL inhibited tumor growth in the treatment group by 78% (171 +/- 52 mm(3)) at d 21 after virus infection compared to the control group (776 +/- 241 mm(3)). Microvessel density was significantly inhibited in tumor tissues treated with rAAV-KAL. rAAV-KAL also decreased the proportion of proliferating cells (Ki-67 positive cells) in tumors compared with the control group. rAAV-mediated expression of KAL inhibits the growth of colon cancer by reducing angiogenesis and proliferation of tumor cells, and may provide a promising anti-angiogenesis-based approach to the treatment of metastatic colorectal cancer.

Astrocyte‐ and hepatocyte‐specific expression of genes from the distal serpin subcluster at 14q32.1 associates with tissue‐specific chromatin structures

The distal serpin subcluster contains genes encoding alpha1-antichymotrypsin (ACT), protein C inhibitor (PCI), kallistatin (KAL) and the KAL-like protein, which are expressed in hepatocytes, but only the act gene is expressed in astrocytes. We show here that the tissue-specific expression of these genes associates with astrocyte- and hepatocyte-specific chromatin structures. In hepatocytes, we identified 12 Dnase I-hypersensitive sites (DHSs) that were distributed throughout the entire subcluster, with the promoters of expressed genes accessible to restriction enzyme digestion. In astrocytes, only six DHSs were located exclusively in the 5' flanking region of the act gene, with its promoter also accessible to restriction enzyme digestion. The acetylation of histone H3 and H4 was found throughout the subcluster in both cell types but this acetylation did not correlate with the expression pattern of these serpin genes. Analysis of histone modifications at the promoters of the act and pci genes revealed that methylation of histone H3 on lysine 4 correlated with their expression pattern in both cell types. In addition, inhibition of methyltransferase activity resulted in suppression of ACT and PCI mRNA expression. We propose that lysine 4 methylation of histone H3 correlates with the tissue-specific expression pattern of these serpin genes.

Kallistatin Inhibits Endothelial Cell Proliferation, Migration and Adhesion in Vitro and Angiogenesis in the Ischemic Hindlimb of Rats

P76 Kallistatin is a serine proteinase inhibitor (serpin) which has multifunctions including regulation of tissue kallikrein activity, blood pressure, inflammation and neointima hyperplasia. In this study, we investigated the potential role of kallistatin in vascular biology by studying its effects on the proliferation, migration and adhesion of cultured primary human endothelial cells in vitro, and angiogenesis in the ischemic hindlimb of rats. Purified kallistatin significantly inhibits cultured endothelial cell proliferation, migration and adhesion induced by VEGF or bFGF. To further investigate the role of kallistatin in vascular growth in vivo, we prepared adenovirus carrying the human kallistatin gene under the control of the cytomegalovirus promoter/enhancer (Ad.CMV-cHKBP). Expression of recombinant human kallistatin in HEK 293 cells transfected with Ad.CMV-cHKBP was identified by a specific ELISA. The effect of adenovirus-mediated kallistatin gene delivery on angiogenesis was evaluated in a rat model of hindlimb ischemia. Adenovirus carrying the human kallistatin or green fluorescent protein (GFP) gene were injected locally into the ischemic adductor at the time of surgery. Histological and morphometric analysis at 14 days post injection showed that adenovirus-mediated kallistatin gene delivery significantly reduced capillary density in the ischemic muscle as compared to that of control rats injected with GFP. The anti-angiogenic effect of kallistatin was associated with reduced regional blood flow in the ischemic hindlimb measured by microsphere assays. Expression of human kallistatin was identified in the injected muscle and immunoreactive human kallistatin levels were measured in the muscle and in the circulation of rats following kallistatin gene delivery. These results demonstrate a novel role of kallistatin in the inhibition of angiogenesis and in vascular remodeling.