Unsaturated or saturated dietary fat-mediated steatosis impairs hepatic regeneration following partial hepatectomy in mice.

BACKGROUND: Partial hepatectomy is a preferred treatment option for many patients with hepatocellular carcinoma however, pre-existing pathological abnormalities originating from hepatic steatosis can alter the decision to perform surgery or postoperative outcomes as a consequence of the impact steatosis has on liver regeneration. AIM: The aim of this study was to investigate the role of a saturated or unsaturated high fat diet-mediated steatosis on liver regeneration following partial hepatectomy. METHODS: Mice were fed a low-fat control diet (CD, 13% fat), lard-based unsaturated (LD, 60% fat) or milk-based saturated high fat diet (MD, 60% fat) for 16 weeks at which time partial hepatectomy (approx. 70% resection) was performed. At days-2 and 7 post hepatectomy, one hour prior to euthanization, mice were injected with 5-bromo-2'-deoxyuridine in order to monitor hepatic regeneration. Serum was collected and assessed for levels of ALT and AST. Resected and regenerated liver tissue were examined for inflammation-indicative markers employing RT-PCR, Western blots, and histological methods. RESULTS: Mice fed LD or MD exhibited higher NAFLD scores, increased expression of inflammatory cytokines, neutrophil infiltration, macrophage accumulation, increased apoptosis, and elevated levels of serum ALT and AST activities, a decrease in the number of BrdU-incorporated-hepatocytes in the regenerated livers compared to the mice fed CD. Mice fed MD showed significantly lower percent of BrdU-incorporated hepatocytes and a higher trend of inflammation compared to the mice fed LD. CONCLUSION: A diet rich in saturated or unsaturated fat results in NASH with decreased hepatic regeneration however unsaturated fat diet cause lower inflammation and higher regeneration than the saturated fat diet following partial hepatectomy in mice.

Use of intra-aortic counterpulsation in cardiogenic shock post-liver transplantation.

Left ventricular dysfunction resulting in cardiogenic shock occurs infrequently following organ reperfusion in liver transplantation. The etiology of the cardiogenic shock is often multifactorial and difficult to manage due to the complex nature of the procedure and the patient's baseline physiology. Traditionally, this hemodynamic instability is managed medically using inotropic agents and vasopressor support. If medical treatment is insufficient, the use of an intra-aortic balloon pump for counterpulsation may be employed to improve the hemodynamics and stabilize the patient. Here, we analyze three cases and review the literature.

Dietary Saturated Fat Promotes Development of Hepatic Inflammation Through Toll-Like Receptor 4 in Mice.

Nonalcoholic steatohepatitis (NASH) is currently the third most common cause of end stage liver disease necessitating transplantation. The question remains how inflammation and NASH develop in the setting of nonalcoholic fatty liver disease (NAFLD) and steatosis. Understand the roles of toll-like receptor 4 (TLR4) and dietary fats in the development of hepatic inflammation. Wild-type and TLR4 KO mice were fed a standard high fat diet (LD), a high saturated fat diet (MD), or an isocaloric control diet (CD). Sera and tissue were analyzed for development of hepatic steatosis, inflammation, and injury. MD induced features of hepatic steatosis and inflammation in wild-type, but not in TLR4 KO, mice. TLR4 KO prevented MD induced increases in NAFLD activity scores, serum alanine aminotransferase levels, and inflammatory cytokine expression. Inflammatory cell infiltration and cytokine expression were also lower in the TLR4 KO mice livers than wild-type mice fed MD. Hepatic expression of Collagen I transcripts and collagen deposition were also decreased in the TLR4 KO MD animals. Results show that TLR4 plays a critical role in the effects of dietary fat composition on the development of hepatic steatosis, inflammation, and injury consistent with nonalcoholic steatohepatitis. J. Cell. Biochem. 117: 1613-1621, 2016. © 2015 Wiley Periodicals, Inc.

Racial disparity outcomes in patients undergoing hepatectomy: is baseline kidney function a potential explanation?

Background-Reasons underlying disparities in outcomes in liver resections between patients who are African American and patients who are not are poorly understood. Methods-An observational longitudinal cohort study was performed. Clinical data were collected from medical records of 166 patients (59 African American, 107 not) undergoing partial hepatectomy between 2004 and 2012. Univariate and multivariate analyses were performed. Results-African Americans patients undergoing partial hepatectomy were more likely to be female, heavier, have hemangiomas or adenomas, and have hepatic steatosis on explant. Intraoperatively, African Americans had longer surgical times, higher estimated blood loss, and greater use of blood products. Major postoperative complications were significantly more common in African Americans. Multivariable modeling demonstrated that race, history of hepatitis C, and estimated blood loss were the only variables that were independently associated with a major complication; however, baseline serum creatinine level was the only variable that significantly modified the effect of race on complications. Conclusions-African Americans with normal serum creatinine levels had a similar rate of complication to patients who were not African American, but as the baseline serum level of creatinine increased, the odds ratio for a complication developing increased dramatically in the African American patients, suggesting that the disparities seen are predominantly driven by a subset of African American patients who have preexisting renal insufficiency.

Development of steatohepatitis in Ob/Ob mice is dependent on Toll-like receptor 4.

BACKGROUND AND AIM: The etiology of non-alcoholic fatty liver disease (NAFLD) progression, and why some patients develop non-alcoholic steatohepatitis (NASH) vs. uncomplicated NAFLD, is not well understood. Obesity and NAFLD are thought to be associated with high circulating levels of leptin; however, the role of leptin in NASH has been controversial. Secondly, as ob/ob mice are known to have elevated circulating levels of TLR4-stimulating endotoxin secondary to increased intestinal permeability. MATERIAL AND METHODS: We evaluated the long-term effects of steatosis on the livers of aleptinemic (OB) mice and the role of TLR4 in the development of hepatic sequelae in these animals. RESULTS: At 20 weeks of age OB animals displayed grossly steatotic livers, but also features of early stage NASH including hepatocellular ballooning and numerous necroinflammatory foci with associated changes in serum aspartate aminotransferase (AST) and alanine transaminase (ALT). TLR4 KO did not affect the development of obesity or steatosis in ob/ob mice, but protected these animals from hepatitis and liver injury. CONCLUSIONS: In conclusion, the data presented here indicate that steatohepatitis develops in the absence of leptin, and that TLR4 is integral to the development NASH secondary to hyperphagia.

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either ß-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium.

A safe and simpler diagnosis of gastric schwannomas.

Although gastric schwannomas usually are nonmalignant, these tumors can undergo malignant transformation. For diagnosis, endoluminal routes are believed to decrease the chance of cancerous cell dissemination. We present a case where a percutaneous route was utilized with supporting evidence for the safe use of this method for diagnosis.

Induction therapy: clinical and quality of life outcomes in aged renal transplant recipients.

BACKGROUND: One primary purpose of transplant is to improve quality of life (QOL) in renal transplant recipients (RTRs) ≥ 50 yr of age, where death with a functioning graft limits life years gained. We aimed to determine the impact of induction therapy, with its subsequent effects on rejection, infection, and readmissions, on QOL. METHODS: Subanalysis of patients ≥ 50 yr of age that participated in a single-center, prospective, risk-stratified, randomized, open-label study. Two hundred RTRs ≥ 50 yr of age. INTERVENTIONS: All patients received either rabbit antithymocyte globulin (rATG) or interleukin 2 receptor antagonists (IL-2RA) in addition to tacrolimus (FK), mycophenolate mofetil (MMF), and corticosteroids in a randomized fashion. Outcome analyses included safety, efficacy, and QOL. RESULTS: Results reported 1 yr post-transplant. Of 111 patients ≥ 50 yr old, 48 received IL-2RA and 63 received rATG. Baseline characteristics were similar between groups. Patients that received rATG had a trend toward lower acute rejection rates, fewer readmissions, and fewer supratherapeutic tacrolimus troughs, with similar rates of infections. QOL analysis demonstrated patients that received rATG were significantly more likely to have improvements in physical and social functioning after transplant. CONCLUSIONS: Contrary to the common practice, T-cell depletion in recipients ≥ 50 yr of age may be beneficial.

Inclusion of dynamic clinical data improves the predictive performance of a 30-day readmission risk model in kidney transplantation.

BACKGROUND: Thirty-day readmissions (30DRA) are a highly scrutinized measure of healthcare quality and relatively frequent among kidney transplants (KTX). Development of predictive risk models is critical to reducing 30DRA and improving outcomes. Current approaches rely on fixed variables derived from administrative data. These models may not capture clinical evolution that is critical to predicting outcomes. METHODS: We directed a retrospective analysis toward: (1) developing parsimonious risk models for 30DRA and (2) comparing efficiency of models based on the use of immutable versus dynamic data. Baseline and in-hospital clinical and outcomes data were collected from adult KTX recipients between 2005 and 2012. Risk models were developed using backward logistic regression and compared for predictive efficacy using receiver operating characteristic curves. RESULTS: Of 1147 KTX patients, 123 had 30DRA. Risk factors for 30DRA included recipient comorbidities, transplant factors, and index hospitalization patient level clinical data. The initial fixed variable model included 9 risk factors and was modestly predictive (area under the curve, 0.64; 95% confidence interval [95% CI], 0.58-0.69). The model was parsimoniously reduced to 6 risks, which remained modestly predictive (area under the curve, 0.63; 95% CI, 0.58-0.69). The initial predictive model using 13 fixed and dynamic variables was significantly predictive (AUC, 0.73; 95% CI, 0.67-0.80), with parsimonious reduction to 9 variables maintaining predictive efficacy (AUC, 0.73; 95% CI, 0.67-0.79). The final model using dynamically evolving clinical data outperformed the model using static variables (P=0.009). Internal validation demonstrated that the final model was stable with minimal bias. CONCLUSIONS: We demonstrate that modeling dynamic clinical data outperformed models using immutable data in predicting 30DRA.

Cardiovascular risk factors contribute to disparities in graft outcomes in African American renal transplant recipients: a retrospective analysis.

Data examining cardiovascular (CV) risk factors in renal transplant recipients (RTRs) and their contribution to the disparity in graft survival between African American (AA) patients and non-AAs is limited. A single-center, retrospective analysis of 1003 adult RTRs from January 1, 2000 to May 1, 2008 to inspect the impact of race on post-transplant CV events, treatment of CV risk factors and their independent influence on graft outcomes was performed. AAs experienced a higher incidence of late graft loss, with 1- and 5-year graft survival rates of 93% and 76% vs 95% and 84% in the non-AA group, respectively. AA patients had a higher prevalence of hypertension (HTN) and diabetes mellitus (DM) and demonstrated reduced control of DM post-transplant (AA 74% vs non-AA 82%, p = 0.053). Multivariate analysis for graft survival indicated acute rejection, delayed graft function (DGF) and incidence of CV events were significant risk factors for graft failure, while the use of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs) and 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors were protective. In conclusion, after controlling for CV risk factors and events, race did not have an independent effect on outcomes, suggesting CV risk factors and events contribute to this disparity. Clinical summary. AAs experienced a higher rate of graft failure and CV events; after adjusting for multiple immunological and CV risk factors, race no longer remained an independent risk factor for post-transplant CV events or graft failure; although disparities in post-transplant outcomes remain, race alone does not account for the disparity; the racial disparity is due to the higher incidence of DGF and acute rejection, as well as traditional CV risk factors, including HTN and DM.

Clinical outcomes and costs associated with in-hospital biliary complications after liver transplantation: a cross-sectional analysis.

INTRODUCTION: In-hospital biliary complications (BCs) after liver transplantation (LT) are reported in up to 20 % of patients and contribute to poor outcomes and increased costs. Existing single-center outcome and cost analyses studies are limited in scope. METHODS: This is a cross-sectional analysis of national data involving 7,967 patients transplanted between 2011 and 2012 with the primary aim of determining the association between BCs and clinical outcomes and costs. Age, race, diagnosis, and severity of illness are associated with the development of BCs. RESULTS: BCs develop in 14.6 % of LT recipients and have substantial implications for perioperative outcomes, including length of hospital and ICU stay (27.9 vs 19.6 mean days, p < 0.001 and 12.0 vs 8.3 mean days, p < 0.001, respectively), in-hospital morbidity (39 vs 27 %, p < 0.001), 30-day readmissions (14.8 vs 11.2 %, p < 0.001), and in-hospital mortality (5.8 vs 4.0 %, p < 0.001). BCs contributed to a mean increase in in-hospital costs of $36,212 (p < 0.001), due to increases in accommodations ($9,539, p < 0.001), surgical services ($3,988, p < 0.001), and pharmacy services ($8,445, p < 0.001). DISCUSSION: BCs are a predominant etiology for in-hospital morbidity and mortality, while contributing significantly to the high cost of LT. Efforts should be focused on understanding salient and modifiable risk factors, while developing innovative strategies to reduce BCs.

Adenovirus-mediated eNOS expression augments liver injury after ischemia/reperfusion in mice.

Hepatic ischemia/reperfusion (l/R) injury continues to be a critical problem. The role of nitric oxide in liver I/R injury is still controversial. This study examines the effect of endothelial nitric oxide synthase (eNOS) over-expression on hepatic function following I/R. Adenovirus expressing human eNOS (Ad-eNOS) was administered by tail vein injection into C57BL/6 mice. Control mice received either adenovirus expressing LacZ or vehicle only. Sixty minutes of total hepatic ischemia was performed 3 days after adenovirus treatment, and mice were sacrificed after 6 or 24 hrs of reperfusion to assess hepatic injury. eNOS over expression caused increased liver injury as evidenced by elevated AST and ALT levels and decreased hepatic ATP content. While necrosis was not pervasive in any group, TUNEL demonstrated significantly increased apoptosis in Ad-eNOS infected livers. Western blotting demonstrated increased levels of protein nitration and upregulation of the pro-apoptotic proteins bax and p53. Our data suggest that over-expression of eNOS is detrimental in the setting of hepatic I/R.

New method of stent-facilitated arterial reconstruction for orthotopic mouse liver transplantation.

BACKGROUND: Arterialized orthotopic liver transplantation (OLT) in the mouse mimics human liver transplantation physiologically and clinically. The present method of sutured anastomosis for reconstruction of the hepatic artery is complex and is associated with high incidence of complications and failure. This makes the endpoint assessment of using this complex model difficult because of the many variables of the technical aspect. METHODS: A total of 14 pairs of donors and recipients from syngeneic male mice were used for arterialized OLT. The grafts were stored in University of Wisconsin solution at 4°C for less than 4 h, and the recipients underwent OLT using a two-cuff technique. The arterial reconstruction was facilitated by the use of a single stent connecting the donor liver artery segment to the recipient common hepatic artery. RESULTS: All 14 recipients survived with the time for arterial reconstruction ranging from 4-10 min. Patency of the artery was confirmed by transecting the artery near the graft 2 and 14 d after transplantation. At day 2, five of the six arteries transected were patent and at day 14, seven of the remaining eight were patent for an overall patency rate of 85.7%. CONCLUSIONS: The stent-facilitated arterial reconstruction can be done quickly with a high patency rate. This model expands the translational research efforts to address marginal livers such as steatotic livers.

Mitochondrial uncoupling protein 2 induces cell cycle arrest and necrotic cell death.

Uncoupling protein 2 (UCP2) is a mitochondrial membrane protein that regulates energy metabolism and reactive oxygen species (ROS) production. We generated mouse carboxy- and amino-terminal green fluorescent protein (GFP)-tagged UCP2 constructs to investigate the effect of UCP2 expression on cell proliferation and viability. UCP2-transfected Hepa 1-6 cells did not show reduced cellular adenosine triphosphate (ATP) but showed increased levels of glutathione. Flow cytometry analysis indicated that transfected cells were less proliferative than nontransfected controls, with most cells blocked at the G1 phase. The effect of UCP2 on cell cycle arrest could not be reversed by providing exogenous ATP or oxidant supply, and was not affected by the chemical uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). However, this effect of UCP2 was augmented by treatment with genistein, a tyrosine kinase inhibitor, which by itself did not affect cell proliferation on control hepatocytes. Western blotting analysis revealed decreased expression levels of CDK6 but not CDK2 and D-type cyclins. Examination of cell viability in UCP2-transfected cells with Trypan Blue and Annexin-V staining revealed that UCP2 transfection led to significantly increased cell death. However, characteristics of apoptosis were absent in UCP2-transfected Hepa 1-6 cells, including lack of oligonucleosomal fragmentation (laddering) of chromosomal DNA, release of cytochrome c from mitochondria, and cleavage of caspase-3. In conclusion, our results indicate that UCP2 induces cell cycle arrest at G1 phase and causes nonapoptotic cell death, suggesting that UCP2 may act as a powerful influence on hepatic regeneration and cell death in the steatotic liver.

Intereukin-10 and Kupffer cells protect steatotic mice livers from ischemia-reperfusion injury.

Steatotic livers are more sensitive to ischemia/reperfusion (I/R) and are thus routinely rejected for transplantation because of their increased rate of primary nonfunction (PNF). Lean livers have less I/R-induced damage and inflammation due to Kupffer cells (KC), which are protective after total, warm, hepatic I/R with associated bowel congestion. This protection has been linked to KC-dependent expression of the potent anti-inflammatory cytokine interleukin-10 (IL-10). We hypothesized that pretreatment with exogenous IL-10 would protect the steatotic livers of genetically obese (ob/ob) mice from inflammation and injury induced by I/R. Lean and ob/ob mice were pretreated with either IL-10 or liposomally-encapsulated bisphosphonate clodronate (shown to deplete KC) prior to total, warm, hepatic I/R. IL-10 pretreatment increased survival of ob/ob animals at 24 hrs post-I/R from 30% to 100%, and significantly decreased serum ALT levels. At six hrs post-I/R, IL-10 pretreatment increased IL-10 mRNA expression, but suppressed up-regulation of the pro-inflammatory cytokine IL-1ß mRNA. However, ALT levels were elevated at six hrs post-I/R in KC-depleted animals. These data reveal that pretreatment with IL-10 protects steatotic livers undergoing I/R, and that phagocytically active KC retain a hepatoprotective role in the steatotic environment.

Efficient method of genotyping ob/ob mice using high resolution melting analysis.

OBJECTIVE: Direct health care costs of obesity continue to grow throughout the world and research on obesity disease models are on the rise. The ob/ob mouse is a well-characterized model of obesity and associated risk factors. Successful breeding and backcrossing onto different backgrounds are essential to create knockout models. Ob/ob mice are sterile and heterozygotes must be identified by genotyping to maintain breeding colonies. Several methods are employed to detect the ob mutant allele, a single nucleotide polymorphism (SNP). Gel based methods are time consuming and inconsistent, and non-gel based assays rely upon expensive and complex reagents or instruments. A fast, high-throughput, cost effective, and consistent method to identify Lep(ob) mutation is much needed. DESIGN AND METHODS: Primers to produce an amplicon for High Resolution Melting Analysis (HRM) of the Lep(ob) SNP were designed and validated. RESULTS: Fluorescence normalized high resolution melting curve plots delineated ob/+, ob/ob, and WT genotypes. Genotypes were also confirmed phenotypically. CONCLUSIONS: HRM of the Lep(ob) SNP allows closed-tube identification of the Lep(ob) mutation using a real-time PCR machine now common to most labs/departments. Advantages of this method include assay sensitivity/accuracy, low cost dyes, less optimization, and cost effectiveness as compared to other genotyping techniques.

Cerulenin blockade of fatty acid synthase reverses hepatic steatosis in ob/ob mice.

Fatty liver or hepatic steatosis is a common health problem associated with abnormal liver function and increased susceptibility to ischemia/reperfusion injury. The objective of this study was to investigate the effect of the fatty acid synthase inhibitor cerulenin on hepatic function in steatotic ob/ob mice. Different dosages of cerulenin were administered intraperitoneally to ob/ob mice for 2 to 7 days. Body weight, serum AST/ALT, hepatic energy state, and gene expression patterns in ob/ob mice were examined. We found that cerulenin treatment markedly improved hepatic function in ob/ob mice. Serum AST/ALT levels were significantly decreased and hepatic ATP levels increased in treated obese mice compared to obese controls, accompanied by fat depletion in the hepatocyte. Expression of peroxisome proliferator-activated receptors α and γ and uncoupling protein 2 were suppressed with cerulenin treatment and paralleled changes in AST/ALT levels. Hepatic glutathione content were increased in some cases and apoptotic activity in the steatotic livers was minimally changed with cerulenin treatment. In conclusion, these results demonstrate that fatty acid synthase blockade constitutes a novel therapeutic strategy for altering hepatic steatosis at non-stressed states in obese livers.

Mitochondrial uncoupling protein-2 deficiency protects steatotic mouse hepatocytes from hypoxia/reoxygenation.

Steatotic livers are sensitive to ischemic events and associated ATP depletion. Hepatocellular necrosis following these events may result from mitochondrial uncoupling protein-2 (UCP2) expression. To test this hypothesis, we developed a model of in vitro steatosis using primary hepatocytes from wild-type (WT) and UCP2 knockout (KO) mice and subjected them to hypoxia/reoxygenation (H/R). Using cultured hepatocytes treated with emulsified fatty acids for 24 h, generating a steatotic phenotype (i.e., microvesicular and broad-spectrum fatty acid accumulation), we found that the phenotype of the WT and UCP2 KO were the same; however, cellular viability was increased in the steatotic KO hepatocytes following 4 h of hypoxia and 24 h of reoxygenation; Hepatocellular ATP levels decreased during hypoxia and recovered after reoxygenation in the control and UCP2 KO steatotic hepatocytes but not in the WT steatotic hepatocytes; mitochondrial membrane potential in WT and UCP2 KO steatotic groups was less than control groups but higher than UCP2 KO hepatocytes. Following reoxygenation, lipid peroxidation, as measured by thiobarbituric acid reactive substances, increased in all groups but to a greater extent in the steatotic hepatocytes, regardless of UCP2 expression. These results demonstrate that UCP2 sensitizes steatotic hepatocytes to H/R through mitochondrial depolarization and ATP depletion but not lipid peroxidation.

STAT3 activation in pressure-overloaded feline myocardium: role for integrins and the tyrosine kinase BMX.

Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.