Syamal D. Bhattacharya

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH‐related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild‐type (WT) mice, Patched‐deficient (Ptc+/−) (overly active Hh signaling) mice, and OPN‐deficient mice before and after feeding methionine and choline–deficient (MCD) diets to induce NASH‐related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc+/− mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN‐deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up‐regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up‐regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte‐derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). Conclusion: OPN is Hh‐regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH (HEPATOLOGY 2011)

Osteopontin promotes CCL5-mesenchymal stromal cell-mediated breast cancer metastasis

The interaction between cancer and its local microenvironment can determine properties of growth and metastasis. A critical component of the tumor microenvironment in this context is the cancer-associated fibroblast (CAF), which can promote tumor growth, angiogenesis and metastasis. It has been hypothesized that CAF may be derived from mesenchymal stromal cells (MSC), derived from local or distant sources. However, the signaling mechanisms by which tumors and MSCs interact to promote CAF-dependent cancer growth are largely unknown. In this study with in vitro and in vivo models using MDA-MB231 human breast cancer cells, we demonstrate that tumor-derived osteopontin (OPN) induces MSC production of CCL5; the mechanism involves OPN binding to integrin cell surface receptors and activator protein-1 c-jun homodimer transactivation. In a murine xenograft model, concomitant inoculation of MSC with MDA-MB231 cells induces: (i) significantly increased growth and metastasis of MB231 cells and (ii) increased MSC migration to metastatic sites in lung and liver; this mechanism is both OPN and CCL5 dependent. MSCs retrieved from sites of metastases exhibit OPN-dependent expression of the CAF markers, α-smooth muscle actin, tenascin-c, CXCL12 (or stromal cell-derived factor 1) and fibroblast-specific protein-1 and the matrix metalloproteinases (MMP)-2 and MMP-9. Based upon these results, we propose that tumor-derived OPN promotes tumor progression via the transformation of MSC into CAF.

Retrograde ascending aortic dissection as an early complication of thoracic endovascular aortic repair

OBJECTIVE Retrograde ascending aortic dissection (rAAD) is a potential complication of thoracic endovascular aortic repair (TEVAR), yet little data exist regarding its occurrence. This study examines the incidence, etiology, and outcome of this event. METHODS A prospective institutional database was used to identify cases of acute rAAD following TEVAR from a cohort of 309 consecutive procedures from March 2005 (date of initial Food and Drug Administration approval) to September 2010. The database was analyzed for the complication of rAAD as well as relevant patient and operative variables. RESULTS The incidence of rAAD was 1.9% (6/309); all cases occurred with proximal landing zone in the ascending aorta and/or arch (zones 0-2). All were identified in the perioperative period (range, 0-6 days) with 33% (2/6) 30-day/in-hospital mortality. Eighty-three percent (5/6) underwent emergent repair; one patient died without repair. rAAD patients were similar to the non-rAAD group (n = 303) across pertinent variables, including age, gender, race, and device size (all P > .1). rAAD incidence by aortic pathology was 1.0% (2/200) for aneurysm, 4.4% (4/91) for dissection, and 0% (0/18) for transection; P = .08. rAAD incidence by device was TAG (Gore) 1.0% (2/205), Talent (Medtronic) 4.7% (2/43), and Zenith TX2 (Cook) 3.6% (2/55). rAAD incidence was observed to be higher among patients with an ascending aortic diameter ≥ 4.0 cm (4.8% vs 0.9% for ascending diameter <4.0 cm); P = .047. Incidence was also higher with proximal landing zone in the native ascending aorta (zone 0) 6.9% (2/29) versus 1.4% for all others (4/280); P = .101. For patients with dissection pathology and an ascending aortic diameter ≥ 4.0 cm, 11% (3/28) suffered rAAD; with the combination of native ascending aorta (zone 0) landing zone measuring ≥ 4.0 cm, the incidence was 25% (2/8). Definitive diagnosis was by computed tomography angiography (n = 1), intraoperative transesophageal echocardiography (n = 3), intraoperative arteriography (n = 1), or postmortem autopsy (n = 1). CONCLUSIONS rAAD is a lethal early complication of TEVAR, which may be more common when treating dissection, with devices utilizing proximal bare springs or barbs for fixation, with native zone 0 proximal landing zone and with ascending aortic diameter ≥ 4 cm. Combinations of these risk factors may be particularly high risk. Intraoperative imaging assessment of the ascending aorta should be conducted following TEVAR to avoid under-recognition. National database reporting of this complication is needed to ensure safety and proper application of emerging TEVAR technology.

Midterm results with thoracic endovascular aortic repair for chronic type B aortic dissection with associated aneurysm.

OBJECTIVE Thoracic endovascular aortic repair for chronic type B aortic dissection with associated descending thoracic aneurysm remains controversial. Concerns include potential ischemic complications due to branch vessel origin from the chronic false lumen and continued retrograde false lumen/aneurysm sac pressurization via fenestrations distal to implanted endografts. The present study examines midterm results with thoracic endovascular aortic repair for chronic (>2 weeks) type B aortic dissection with associated aneurysm to better understand the potential role of thoracic endovascular aortic repair for this condition. METHODS Between March 2005 and December 2009, 51 thoracic endovascular aortic repair procedures were performed at a single institution for management of chronic type B dissection. The indication for thoracic endovascular aortic repair was aneurysm in all cases. A subset of 7 patients (14%) underwent placement of the EndoSure wireless pressure measurement system (CardioMEMS, Inc, Atlanta, Ga) in the false lumen adjacent to the primary tear for monitoring aneurysm sac/false lumen pulse pressure after thoracic endovascular aortic repair. RESULTS Mean patient age was 57±12 years (range, 30-82 years); 14 patients (28%) were female. Mean aortic diameter was 6.2±1.4 cm. There were no in-hospital/30-day deaths, strokes, or permanent paraplegia/paresis. There were no complications related to compromise of downstream branch vessels arising from the false lumen. Two patients (3.9%) who had preexisting ascending aortic dilation had retrograde acute type A aortic dissection; both were repaired successfully. Median postoperative length of stay was 4 days. Mean follow-up is 27.0±16.5 months (range, 2-60 months). Actuarial overall survival is 77.7% at 60 months with an actuarial aorta-specific survival of 98% over this same time period. Actuarial freedom from reintervention is 77.3% at 60 months. All patients with the EndoSure wireless pressure measurement system exhibited a decrease in aneurysm sac/false lumen pulse pressure indicating a depressurized false lumen. The aneurysm sac/false lumen pulse pressure ratio decreased from 52%±27% at the predischarge measurement to 14%±5% at the latest follow-up reading (P=.029). CONCLUSIONS Thoracic endovascular aortic repair for chronic type B dissection with associated aneurysm is safe and effective at midterm follow-up. Aneurysm sac/false lumen pulse pressure measurements demonstrate a significant reduction in false lumen endotension, thus ruling out clinically significant persistent retrograde false lumen perfusion and provide proof of concept for a thoracic endovascular aortic repair-based approach. Longer-term follow-up is needed to determine the durability of thoracic endovascular aortic repair for this aortic pathology.

Results With a Selective Revascularization Strategy for Left Subclavian Artery Coverage During Thoracic Endovascular Aortic Repair

BACKGROUND The need for routine left subclavian artery (LSCA) revascularization when this vessel is covered during thoracic endovascular aortic repair remains controversial. We report our results with a selective LSCA revascularization strategy during thoracic endovascular aortic repair. METHODS Between May 2002 and March 2010, 287 thoracic endovascular aortic repair procedures were performed at our institution. LSCA coverage occurred in 145 (51%), which form the basis of this report. RESULTS Left subclavian artery revascularization was performed in 32 patients (22%) through a left common carotid-LSCA bypass. Indications for selective LSCA revascularization included spinal cord protection in 10, patent pedicled left internal mammary artery graft in 9, left arm ischemia after LSCA coverage in 5, origin of the left vertebral artery from the arch in 4, dialysis access in the left arm in 2, and vertebrobasilar insufficiency in 2. There were no instances of dominant left vertebral artery. The revascularized and non-revascularized groups had similar rates of death (6.3% vs 1.8%; p=0.21), stroke (3.1% vs 3.5%; p>0.99), permanent paraplegia or paraparesis (3.1% vs 0%; p=0.22), and type II endoleak (4.3% vs 6.5%; p>0.99). There were no instances of ischemic stroke related to left posterior circulation hypoperfusion. Four complications of carotid-subclavian bypass occurred in 3 patients (9.4%). CONCLUSIONS Selective LSCA revascularization is safe and does not appear to increase the risk of neurologic events. Further, subclavian revascularization is not without complications, which should be considered with regards to a nonselective revascularization strategy.

Intraoperative use of low-dose recombinant activated factor VII during thoracic aortic operations.

BACKGROUND Numerous studies have supported the effectiveness of recombinant activated factor VII (rFVIIa) for the control of bleeding after cardiac procedures; however safety concerns persist. Here we report the novel use of intraoperative low-dose rFVIIa in thoracic aortic operations, a strategy intended to improve safety by minimizing rFVIIa exposure. METHODS Between July 2005 and December 2010, 425 consecutive patients at a single referral center underwent thoracic aortic operations with cardiopulmonary bypass (CPB); 77 of these patients received intraoperative low-dose rFVIIa (≤60 μg/kg) for severe coagulopathy after CPB. Propensity matching produced a cohort of 88 patients (44 received intraoperative low-dose rFVIIa and 44 controls) for comparison. RESULTS Matched patients receiving intraoperative low-dose rFVIIa got an initial median dose of 32 μg/kg (interquartile range [IQR], 16-43 μg/kg) rFVIIa given 51 minutes (42-67 minutes) after separation from CPB. Patients receiving intraoperative low-dose rFVIIa demonstrated improved postoperative coagulation measurements (partial thromboplastin time 28.6 versus 31.5 seconds; p=0.05; international normalized ratio, 0.8 versus 1.2; p<0.0001) and received 50% fewer postoperative blood product transfusions (2.5 versus 5.0 units; p=0.05) compared with control patients. No patient receiving intraoperative low-dose rFVIIa required postoperative rFVIIa administration or reexploration for bleeding. Rates of stroke, thromboembolism, myocardial infarction, and other adverse events were equivalent between groups. CONCLUSIONS Intraoperative low-dose rFVIIa led to improved postoperative hemostasis with no apparent increase in adverse events. Intraoperative rFVIIa administration in appropriately selected patients may correct coagulopathy early in the course of refractory blood loss and lead to improved safety through the use of smaller rFVIIa doses. Appropriately powered randomized studies are necessary to confirm the safety and efficacy of this approach.

Predictors of electrocerebral inactivity with deep hypothermia

OBJECTIVE Cooling to electrocerebral inactivity (ECI) by electroencephalography (EEG) remains the gold standard to maximize cerebral and systemic organ protection during deep hypothermic circulatory arrest (DHCA). We sought to determine predictors of ECI to help guide cooling protocols when EEG monitoring is unavailable. METHODS Between July 2005 and July 2011, 396 patients underwent thoracic aortic operation with DHCA; EEG monitoring was used in 325 (82%) of these patients to guide the cooling strategy, and constituted the study cohort. Electroencephalographic monitoring was used for all elective cases and, when available, for nonelective cases. Multivariable linear regression was used to assess predictors of the nasopharyngeal temperature and cooling time required to achieve ECI. RESULTS Cooling to a nasopharyngeal temperature of 12.7°C or for a duration of 97 minutes was required to achieve ECI in >95% of patients. Only 7% and 11% of patients achieved ECI by 18°C or 50 minutes of cooling, respectively. No independent predictors of nasopharyngeal temperature at ECI were identified. Independent predictors of cooling time included body surface area (18 minutes/m(2)), white race (7 minutes), and starting nasopharyngeal temperature (3 minutes/°C). Low complication rates were observed (ischemic stroke, 1.5%; permanent paraparesis/paraplegia, 1.5%; new-onset dialysis, 2.2%; and 30-day/in-hospital mortality, 4.3%). CONCLUSIONS Cooling to a nasopharyngeal temperature of 12.7°C or for a duration of 97 minutes achieved ECI in >95% of patients in our study population. However, patient-specific factors were poorly predictive of the temperature or cooling time required to achieve ECI, necessitating EEG monitoring for precise ECI detection.

Predictors of massive transfusion with thoracic aortic procedures involving deep hypothermic circulatory arrest

OBJECTIVE Massive perioperative blood product transfusion may be required with thoracic aortic operations and is associated with poor outcomes. We analyzed independent predictors of massive transfusion in thoracic aortic surgical patients undergoing deep hypothermic circulatory arrest. METHODS The study consisted of 168 consecutive patients undergoing open thoracic aortic procedures involving deep hypothermic circulatory arrest between July 2005 and August 2008. We identified 26 preoperative and procedural variables as potentially related to blood product use, tested for association with total blood products transfused by multivariate linear regression model, and constructed logistic regression model for massive transfusion (requiring ≥ 5 units of transfused packed red blood cells between incision and 48 postoperative hours). RESULTS Multivariate linear regression determined that 6 significant variables accounted for 42% of variation in total blood products transfused: age (P = .008), preoperative hemoglobin (P = .04), weight (P = .02), cardiopulmonary bypass time (P < .0001), emergency status (P < .0001), and resternotomy (P < .0001). Final predictive logistic regression model included 1-g/dL increase in preoperative hemoglobin (odds ratio, 0.54; 95% confidence interval, 0.43-0.69; P < .0001), 10-minute increase in cardiopulmonary bypass time (odds ratio, 1.15; 95% confidence interval, 1.05-1.26; P = .0026), and emergency status (odds ratio, 4.02; 95% confidence interval, 1.53-10.55; P = .0047. CONCLUSIONS Cardiopulmonary bypass time, emergency status, and preoperative hemoglobin were independent predictors of massive transfusion. These variables, along with weight, age, and resternotomy, were associated with total blood product use in thoracic aortic operations involving deep hypothermic circulatory arrest.

Cardiac catheterization within 1 to 3 days of proximal aortic surgery is not associated with increased postoperative acute kidney injury

OBJECTIVE Cardiac catheterization shortly before coronary artery bypass grafting or valve surgery has been associated with increased postoperative acute kidney injury. The relationship between catheterization timing and acute kidney injury after proximal aortic surgery remains unknown. METHODS Between August 2005 and February 2011, a total of 285 consecutive patients underwent cardiac catheterization before elective proximal aortic surgery with cardiopulmonary bypass at a single institution. The association between timing of catheterization and postoperative acute kidney injury (defined as postoperative increase in serum creatinine ≥ 50% of baseline) was assessed using logistic regression analysis. RESULTS Of 285 patients, 152 (53%) underwent catheterization on preoperative days 1 to 3 and 133 (47%) underwent catheterization on preoperative day 4 or before. Acute kidney injury occurred in 88 (31%) patients, 3 (1.1%) requiring dialysis. Acute kidney injury occurred in 37 (24%) patients catheterized on preoperative days 1 to 3, and 51 (38%) patients catheterized on preoperative day 4 or before. Catheterization on preoperative days 1 to 3 was not associated with an increased risk of acute kidney injury relative to catheterization on preoperative day 4 or before (unadjusted odds ratio, 0.52; 95% confidence interval, 0.31-0.86; P = .01; adjusted odds ratio, 0.35; 95% confidence interval, 0.17-0.73; P = .005). CONCLUSIONS Cardiac catheterization within 1 to 3 days of elective proximal aortic surgery appears safe and should be considered acceptable practice for patients at low risk of acute kidney injury.

Osteopontin and protein kinase C regulate PDLIM2 activation and STAT1 ubiquitination in LPS-treated murine macrophages.

The molecular pathways regulating signal transducer and activator of transcription 1 (STAT1) levels in states of inflammation are incompletely understood. The suppressor of cytokine signaling, protein inhibitor of STAT, and SHP-1/2 tyrosine phosphatases ultimately regulate activity of STAT molecules. However, these mechanisms do not degrade STAT proteins. In this regard, using a murine macrophage model of LPS stimulation, we previously demonstrated that osteopontin (OPN) increased STAT1 ubiquitination and 26 S proteasome degradation via the ubiquitin E3 ligase, PDLIM2. In this study, we further characterize OPN-dependent activation of PDLIM2 in a model of LPS-stimulated RAW264.7 murine macrophages. We identify serine 137 as a protein kinase C-phosphorylation site in PDLIM2 that is required for ubiquitination of STAT1. PDLIM2 phosphorylation requires OPN expression. Using phospho-mutants and phospho-mimetic constructs of PDLIM2, our in vivo and in vitro ubiquitination studies confirm the role of PDLIM2 in formation and degradation of Ub-STAT1. The functional consequences of PDLIM2-mediated STAT1 degradation were confirmed using an IFN-γ-regulated transcription factor STAT1α reporter construct and chromatin immunoprecipitation assay for the inducible nitric-oxide synthase promoter. In a murine cecal ligation and puncture model of sepsis in wild-type and OPN (−/−) animals, OPN was necessary for PDLIM2 serine phosphorylation and STAT1 ubiquitination in bone marrow macrophages. We conclude that OPN and PDLIM2 are important regulators of STAT1-mediated inflammatory responses.