Richard S. Vander Heide

Transforming Growth Factor-β Stimulates Intestinal Epithelial Focal Adhesion Kinase Synthesis via Smad- and p38-Dependent Mechanisms

Focal adhesion kinase (FAK) regulates cell migration, proliferation, and apoptosis. FAK protein is reduced at the edge of migrating gut epithelial sheets in vitro, but it has not been characterized in restitutive gut mucosa in vivo. Here we show that FAK and activated phospho-FAK (FAK(397)) immunoreactivity was lower in epithelial cells immediately adjacent to human gastric and colonic ulcers in vivo, but dramatically increased in epithelia near the ulcers, possibly reflecting stimulation by growth factors absent in vitro. Transforming growth factor (TGF)-beta, but not fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor, increased FAK levels in Caco-2 and IEC-6 cells. Epithelial immunoreactivity to TGF-beta and phospho-Smad3 was also higher near the ulcers, varying in parallel with FAK. The TGF-beta receptor antagonist SB431542 completely blocked TGF-beta-induced Smad2/3 and p38 activation in IEC-6 cells. SB431542, the p38 antagonist SB203580, and siRNA-mediated reduction of Smad2 and p38alpha prevented TGF-beta stimulation of both FAK transcription and translation (as measured via a FAK promoter-luciferase construct). FAK(397) levels were directly related to total FAK protein expression. Although gut epithelial motility is associated with direct inhibition of FAK protein adjacent to mucosal wounds, TGF-beta may increase FAK protein near but not bordering mucosal ulcers via Smad2/3 and p38 signals. Our results show that regulation of FAK expression may be as important as FAK phosphorylation in critically influencing gut epithelial cell migration after mucosal injury.

Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms

BackgroundThe infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression.ResultsWe analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. HOXA4 was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (P < 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The HOXA4 transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (P < 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (P < 0.0007).ConclusionsOur results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of HOXA4 expression was associated with AAAs, an important aortic disease of the ageing population.

Disseminated Cytomegalovirus Disease in Hosts without Acquired Immunodeficiency Syndrome and without an Organ Transplant

We describe 7 histologically proven cases of cytomegalovirus disease in patients without human immunodeficiency virus and without organ transplants, all of whom had associated comorbid conditions. Therapy with ganciclovir generally resulted in a favorable outcome.

Heat stress activates AKT via focal adhesion kinase-mediated pathway in neonatal rat ventricular myocytes.

Heat stress (HS)-induced cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) to determine whether myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. After HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively with an adenoviral vector. Cell signaling was analyzed with Western blot analysis, while oncosis/apoptosis was assayed by measuring Trypan blue exclusion and/or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM before CI. HS increased association between FAK and phosphatidylinositol 3-kinase as well as causing a significant increase in AKT activity. Increased expression of wild-type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time 0 and after 10 min of CI compared with myocytes expressing empty virus. We conclude that myocardial stress activates cytoskeleton-based signaling pathways that are associated with protection from lethal cell injury.

Aqueous Oxygen Attenuation of Reperfusion Microvascular Ischemia in a Canine Model of Myocardial Infarction

Uncorrected microvascular ischemia may contribute to left ventricular impairment during reperfusion after prolonged coronary artery occlusion. Attenuation of such ischemia in microvessels with impaired erythrocyte flow may require delivery of oxygen at high levels in plasma. Intraarterial infusion of aqueous oxygen (AO) can be used in a site specific manner to achieve hyperoxemic levels of oxygenation in the perfusate. With this new approach, the hypothesis was tested that reperfusion microvascular ischemia can be attenuated.After a 90 min coronary balloon occlusion in a canine model, AO hyperoxemic intracoronary perfusion was performed for 90 min after a 30 min period of autoreperfusion. Control groups consisted of normoxemic reperfusion, both passive (autoreperfusion) and active (roller pump). A significant improvement in left ventricular ejection fraction (p < 0.05) at 2 hr of reperfusion was noted only in the AO hyperoxemia group (17 ± 6% by two dimensional echo), without a significant reduction in the improvement 1 hr after termination of treatment. During AO hyperoxemic perfusion, ECG ST segment isoelectric deviation normalized, and frequency of ventricular premature contractions was significantly reduced, in contrast to the autoreperfusion control group (p < 0.05). Microvascular blood flow, measured as the ischemic/normal left ventricular segment ratio by radiolabeled microspheres immediately after AO hyperoxemic perfusion, was double the value of the autoreperfusion control group at 2 hr of reperfusion (p < 0.05).We conclude that reperfusion microvascular ischemia is attenuated by intracoronary AO hyperoxemic perfusion and acutely improves left ventricular function in this model.

Ischemic preconditioning and heat shock activate Akt via a focal adhesion kinase-mediated pathway in Langendorff-perfused adult rat hearts

Heat stress (HS)-induced cardioprotection is associated with the activation of focal adhesion kinase (FAK) and protein kinase B (Akt) in neonatal rat ventricular myocytes (NRVMs), suggesting that stress-induced activation of survival pathways may be important in protecting intact hearts from irreversible injury. The purposes of this study were 1) to examine the subcellular signaling pathways activated by HS and ischemic preconditioning (IP) in intact hearts, 2) to determine whether HS and IP activate an integrated survival pathway similar to that activated by HS in cultured NRVMs, and 3) to determine whether HS and IP reduce lethal cell injury in perfused intact hearts. Adult rat hearts perfused in the Langendorff mode were subjected to 25 min of global ischemia and 30 min of reperfusion (I/R) either 24 h after whole animal HS or following a standard IP protocol. Myocardial signaling was analyzed using Western blot analysis, whereas cell death was assayed by measuring lactate dehydrogenase release into the perfusate and confirmed by light microscopy. Similar to NRVMs, HS performed in the whole animal 24 h before I/R increased phosphorylation of FAK at tyrosine-397 and protein kinase B (Akt) and resulted in protection from cell death. Using IP as a myocardial stress also resulted in an increased phosphorylation/activation of both FAK and Akt and resulted in reduced cell death in adult perfused rat hearts subjected to I/R. In conclusion, 1) myocardial stress caused by whole animal HS activates cytoskeletal-based survival signaling pathways in whole heart tissue and reduces lethal I/R injury and 2) IP activates the same stress-induced survival pathway and the activation correlates with the well-known cardioprotective effect of IP on lethal I/R injury.

Role of Altered Energy Metabolism in Ischemic Preconditioning

Myocardium that is exposed to a mild, sublethal stress is capable of undergoing adaptive changes that enable it to better tolerate a more severe, potentially lethal exposure to the same (or different) form of stress. This response may be rapid (minutes) or delayed (hours). For example, when animals are exposed to heat stress, the myocardium responds by synthesizing a family of heat shock proteins (HSPs) [1,2], which, 24 hours later, are associated with an increased tolerance to various insults, including heat stress and ischemia [3, 4, 5, 6] (see Chapter 6).

Causes of isolated aortic insufficiency in an urban population in the 1990s a review of 56 surgical pathology cases.

Until recently, the cause of isolated aortic insufficiency (AI) was usually thought to be inflammatory or rheumatic in most cases. However, at our institution we have noted a high prevalence of myxomatous degeneration (MD) in aortic valves removed for AI. In this study we report anatomic observations on valves from 56 consecutive patients with isolated AI undergoing aortic valve replacement surgery. Fifty-six consecutive aortic valves removed at our institution from 1994 to 1996 for isolated AI and/or aortic aneurysm were reviewed. Anatomic features were compared with clinical history and echocardiographic data. The anatomic results were also compared to 22 age-matched control aortic valves obtained at autopsy. In 13/56 cases (23%), a specific valvular cause of AI was determined (infectious endocarditis, seven cases; chronic rheumatic disease, four cases; congenital bicuspid valve, two cases). Of the remaining (idiopathic) 43 cases, 18 (42%) had severe isolated MD defined as >50% expansion of the spongiosa and disruption of the fibrosa by the deposition of acid mucopolysaccharides in the absence of severe calcification, fibrosis, or other pathologic findings. Only 1/22 aortic valves from the autopsy controls had severe MD. Eighteen of the 56 patients also had a clinical history of aortic dilatation/aneurysm of which 12 were confirmed to be dilated by echocardiographic criteria. Of these 12, five (42%) had MD of the aortic valve only, three (25%) had both MD and cystic medial degeneration (CMD) of the aorta, two (17%) had CMD of the aorta only, and two (17%) had no specific diagnosis. Isolated MD of the aortic valve is the most common cause of isolated AI in our patient population. Furthermore, in a subset of non-Marfans patients with both AI and dilatation of the aortic root/aortic aneurysm the incidence of MD is even higher (67%). These results suggest that there is overlap between MD and CMD in non-Marfans patients and that both entities may be part of a spectrum of a generalized connective tissue disorder.

Adenosine therapy at reperfusion and myocardial infarct size

w x We recently reported in this journal 1 that intravenous administration of adenosine, given immediately prior to and continuing for 2.5 h into reperfusion, failed to limit infarct size in an open-chest canine model of regional myocardial infarction. Our study was designed to investigate the ability of adenosine, one putative inhibitor of lethal reperfusion injury, to limit myocardial necrosis following a regional ischemic insult. Both lethal and nonlethal reperfusion injury have been hypothesized to exist. There is strong evidence that myocardial stunning occurs, at least in part, through oxygen-derived free radicals generated upon the onset of arterial reperfusion and therefore stunning is generally accepted to be a form of non-lethal w x reperfusion injury 2 . However, the existence of lethal reperfusion injury is more controversial. The experimental protocol used in our study included therapy with adenosine, based on the dose, route, and timing of administration used in the study of Pitarys et al. w x Ž 3 . These investigators had reported a positive effect i.e., . limitation of infarct size of therapy in a closed-chest canine model. In addition, our experimental protocol was designed to reproduce the results previously reported by Homeister et al. who showed that adenosine plus bolus administration of lidocaine, but not adenosine alone, resulted in cardioprotection in a canine model of regional w x infarction 4 . We were unable to show any limitation of infarct size as a result of intravenous adenosine administration, with or without lidocaine, in our open-chest model. Our negative results were discordant with the reports of marked limitation of infarct size in studies by Forman and w x colleagues 3 . Both openand closed-chest models have been used successfully in many different experimental laboratories to w x study the pathophysiology of ischemia and reperfusion 5 .

Percutaneous Pulmonary Artery Gene Transfer Using Naked Plasmid DNA |[bull]| 120

There are no published reports of successful percutaneous in vivo gene delivery to the pulmonary vascular bed without the use of adjunctive agents. The purpose of our study was to demonstrate the feasibility of recombinant reporter gene transfer to the pulmonary artery of pigs using naked plasmid DNA delivered via percutaneous catheter techniques involving angioplasty. Varying quantities of naked plasmid DNA containing the E. coli lacZ gene in a eukaryotic expression vector were used to transfect pigs that were 10kg to 20kg in weight. One technique for delivery of reporter gene to the pulmonary artery involved pressing plasmid DNA into the vessel wall during angioplasty using a standard angioplasty balloon which was coated with a solution of DNA mixed with heparin. The other delivery technique involved infusing DNA between an angioplasty balloon and a second, surrounding, perforated balloon. We allowed three days for gene expression and then harvested the pulmonary arteries. RNA from the pulmonary arteries was reverse transcribed, and cDNA was amplified by polymerase chain reaction using primers specific for lacZ. Foreign protein expression was evaluated with immunohistochemistry using a polyclonal antibody to β-galactosidase. Successful transcription and translation were determined for both delivery techniques in the treated but not contralateral pulmonary arteries. We conclude that pulmonary artery gene transfer using naked plasmid DNA delivered via percutaneous angioplasty techniques is feasible. The use of naked plasmid DNA removes the potential for toxicity associated with an adjunctive agent such as liposome or adenovirus. Additionally, ours is the first report of gene delivery to the pulmonary vascular bed using angioplasty. The development of this technique provides a novel method for the study of pulmonary vascular biology in vivo and establishes the feasibility of gene insertion into the pulmonary arteries.