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Jahar Bhattacharya

Publications and source records attributed to Jahar Bhattacharya.

At least 19 recordsLinked to original sources
PubMed2006

Real-time lung microscopy.

Although proinflammatory cell signaling in the alveolo-capillary region predisposes to acute lung injury, key cell-signaling mechanisms remain inadequately understood. Alveolo-capillary inflammation is likely to involve coordinated signaling among cells of different phenotypes. For example, migration of inflammatory cells into the alveolus might entail coordinated signaling between adjoining alveolar epithelial and microvascular endothelial cells. The popular cultured cell experimental strategy fails to replicate this multicellular environment. Cultured lung cells, both alveolar and endothelial, undergo phenotypic transformations; hence they might inadequately reflect innate responses of native cells. Consequently, new approaches are required for the investigation of cell signaling in the native setting. Here we summarize new developments in classical intravital microscopy and discuss real-time fluorescence imaging as a novel technique for studying second-messenger mechanisms in the alveolo-capillary region.

Animals↗
PubMed2006

Resealing of endothelial junctions by focal adhesion kinase.

Endothelial cell (EC) junctions determine vascular barrier properties and are subject to transient opening to allow liquid flux from blood to tissue. Although EC junctions open in the presence of permeability-enhancing factors, including oxidants, the mechanisms by which they reseal remain inadequately understood. To model opening and resealing of EC junctions in the presence of an oxidant, we quantified changes in H(2)O(2)-induced transendothelial resistance (TER) in monolayers of rat lung microvascular EC. During a 30-min exposure, H(2)O(2) (100 microM) decreased TER for an initial approximately 10 min, indicating junctional opening. Subsequently, despite continuous presence of H(2)O(2), TER recovered to baseline, indicating the activation of junctional resealing mechanisms. These bimodal TER transients matched the time course of loss and then gain of E-cadherin at EC junctions. The timing of the TER decrease matched the onset of focal adhesion formation, while F-actin increase at the cell periphery occurred with a time course that complemented the recovery of peripheral E-cadherin. In monolayers expressing a focal adhesion kinase (FAK) mutant (del-FAK) that inhibits FAK activity, the initial H(2)O(2)-induced junctional opening was present, although the subsequent junctional recovery was blocked. Expression of transfected E-cadherin was evident at the cell periphery of wild-type but not del-FAK-expressing EC. E-cadherin overexpression in del-FAK-expressing EC failed to effect major rescue of the junctional resealing response. These findings indicate that in oxidant-induced EC junction opening, FAK plays a critical role in remodeling the adherens junction to reseal the barrier.

Adherens Junctions↗
PubMed2006

Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries.

Acute lung injury (ALI), which is associated with a mortality of 30-40%, is attributable to inflammation that develops rapidly across the lung's vast vascular surface, involving an entire lung or even both lungs. No specific mechanism explains this extensive inflammatory spread, probably because of the lack of approaches for detecting signal conduction in lung capillaries. Here, we addressed this question by applying the photolytic uncaging approach to induce focal increases in Ca2+ levels in targeted endothelial cells of alveolar capillaries. Uncaging caused Ca2+ levels to increase not only in the targeted cell, but also in vascular locations up to 150 microm from the target site, indicating that Ca2+ was conducted from the capillary to adjacent vessels. No such conduction was evident in mouse lungs lacking endothelial connexin 43 (Cx43), or in rat lungs in which we pretreated vessels with peptide inhibitors of Cx43. These findings provide the first direct evidence to our knowledge that interendothelial Ca2+ conduction occurs in the lung capillary bed and that Cx43-containing gap junctions mediate the conduction. A proinflammatory effect was evident in that induction of increases in Ca2+ levels in the capillary activated expression of the leukocyte adherence receptor P-selectin in venules. Further, peptide inhibitors of Cx43 completely blocked thrombin-induced microvascular permeability increases. Together, our findings reveal a novel role for Cx43-mediated gap junctions, namely as conduits for the spread of proinflammatory signals in the lung capillary bed. Gap junctional mechanisms require further consideration in the understanding of ALI.

Animals↗
PubMed2006

Lung surfactant secretion by interalveolar Ca2+ signaling.

Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether interalveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca2+ uncaging in intact alveoli of the isolated, blood-perfused rat lung. We loaded alveolar cells with the Ca2+ cage o-nitrophenyl EGTA-AM (NP-EGTA-AM) together with the fluorophores, fluo 4, or LysoTracker green (LTG) to determine, respectively, the cytosolic Ca2+ concentration ([Ca2+]cyt) or type 2 cell secretion. To uncage Ca2+ from NP-EGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased, whereas LTG fluorescence decreased, in the photo-targeted region, indicating that uncaging both increased [Ca2+]cyt and induced secretion. Concomitantly, [Ca2+]cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of interalveolar communication. These conducted responses were inhibited by pretreating alveoli with the connexin43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted [Ca2+]cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, interalveolar Ca2+ signals regulate type 2 cell secretion in adjacent alveoli. Such interalveolar communication might facilitate acinar coordination of alveolar function.

Aniline Compounds↗
PubMed2006

Cell signaling underlying the pathophysiology of pneumonia.

The symposium addressed the burgeoning interest in fundamental mechanisms underlying the onset of pneumonia. Bacteria exploit the lung's innate immune mechanism, resulting in pathophysiological cell signaling. As a consequence inflammation develops, leading to pneumonia. New mechanisms have been identified by which bacteria or bacterial products in the airway induce cross-compartmental signaling that leads to inflammatory consequences. The speakers addressed activation of the transcription factor, NF-kappaB occurring as a consequence of bacterial interactions with specific receptors, such as the Toll-like receptors and the TNF receptor 1 (Prince), or as a consequence of cytokine induction (Mizgerd). Also considered were mechanisms of bacterial virulence in the clinical setting (Wiener-Kronish) and the role of alveolar-capillary signaling mechanisms in the initiation of lung inflammation.

Cell Communication↗
PubMed2005

Inhibition of acid-induced lung injury by hyperosmolar sucrose in rats.

RATIONALE: Acid aspiration causes acute lung injury (ALI). Recently, we showed that a brief intravascular infusion of hyperosmolar sucrose, given concurrently with airway acid instillation, effectively blocks the ensuing ALI. OBJECTIVES: The objective of the present study was to determine the extent to which intravascular infusion of hyperosmolar sucrose might protect against acid-induced ALI when given either before or after acid instillation. METHODS: Our studies were conducted in anesthetized rats and in isolated, blood-perfused rat lungs. We instilled HCl through the airway, and we quantified lung injury in terms of the extravascular lung water (EVLW) content, filtration coefficient (Kfc), and cell counts and protein concentration in the bronchoalveolar lavage. We infused hyperosmolar sucrose via the femoral vein. RESULTS: In anesthetized rats, airway HCl instillation induced ALI as indicated by a 52% increase of EVLW and a threefold increase in Kfc. However, a 15-min intravenous infusion of hyperosmolar sucrose given up to 1 h before or 30 min after acid instillation markedly blunted the increases in EVLW, as well as the increases in cell count, and in protein concentration in the bronchoalveolar lavage. Hyperosmolar pretreatment also blocked the acid-induced increase of Kfc. Studies in isolated perfused lungs indicated that the protective effect of hyperosmolar sucrose was leukocyte independent. CONCLUSIONS: We conclude that a brief period of vascular hyperosmolarity protects against acid-induced ALI when the infusion is administered shortly before, or shortly after, acid instillation in the airway. The potential applicability of hyperosmolar sucrose in therapy for ALI requires consideration.

Animals↗
PubMed2005

Pressure-induced leukocyte margination in lung postcapillary venules.

Although pressure elevation in lung postcapillary venules increases endothelial P-selectin expression, the extent to which P-selectin causes lung leukocyte margination remains controversial. To address this issue, we optically viewed postcapillary venules of the isolated blood-perfused rat lung by real-time fluorescence imaging. To determine leukocyte margination in single postcapillary venules, we quantified the fluorescence of leukocytes labeled in situ with rhodamine 6G (R6G). Although baseline fluorescence was sparse, a 10-min pressure elevation by 10 cmH(2)O markedly increased R6G fluorescence. Both stopping blood flow during pressure elevation and eliminating leukocytes from the perfusion blocked the fluorescence increase, affirming that these fluorescence responses were attributable to pressure-induced leukocyte margination. A P-selectin-blocking MAb and the L- and P-selectin blocker fucoidin each inhibited the fluorescence increase, indicating that P-selectin was critical for inducing margination. Time-dependent imaging of blood-borne fluorescent beads revealed reduction of plasma velocity during pressure elevation. After pressure returned to baseline, a similar reduction of plasma velocity, established by manually decreasing the perfusion rate, prolonged margination. Our findings show that in lung postcapillary venules, the decrease in plasma velocity critically determines pressure-induced leukocyte margination.

Animals↗
PubMed2004

Ongoing angiogenesis in blood vessels of the abdominal aortic aneurysm.

Pathogenesis of the abdominal aortic aneurysm has been attributed to neovascularization of the aortic wall. However, it is not clear whether angiogenesis persists in the aneurysm. In sections of aneurysms, we determined the immunohistochemical distributions of the avb3 integrin, tenascin and endothelial nitric oxide synthase (eNOS), which are markers respectively, of angiogenesis, matrix remodeling and vasoregulatory function. In addition, we used reverse transcription followed by in situ PCR, to determine the distribution of av mRNA. All aneurysm specimens exhibited extensive increases of wall vascularization as compared with the control aortic wall, and showed the presence of perivascular inflammatory exudates containing macrophages and lymphocytes. The neovascularization consisted of thick-walled vessels in the media and adventitia, and capillaries in the subintima. The majority of vessels stained positively for the avb3 antigen and eNOS. Tenascin was deposited as bands that circumscribed thick-walled vessels. The distribution of av mRNA was extensive and was positive even in those vessels that failed to stain for the avb3 protein. No staining was evident in control aortas for the avb3 antigen, tenascin or av mRNA. The upregulation of av mRNA and the avb3 integrin in blood vessels surrounded by a matrix expressing tenascin, indicates that angiogenesis is an ongoing process in the mature aortic aneurysm.

Adult↗
PubMed2004

Cell-cell interactions in regulating lung function.

Tight junction barrier formation and gap junctional communication are two functions directly attributable to cell-cell contact sites. Epithelial and endothelial tight junctions are critical elements of the permeability barrier required to maintain discrete compartments in the lung. On the other hand, gap junctions enable a tissue to act as a cohesive unit by permitting metabolic coupling and enabling the direct transmission of small cytosolic signaling molecules from one cell to another. These components do not act in isolation since other junctional elements, such as adherens junctions, help regulate barrier function and gap junctional communication. Some fundamental elements related to regulation of pulmonary barrier function and gap junctional communication were presented in a Featured Topic session at the 2004 Experimental Biology Conference in Washington, DC, and are reviewed in this summary.

Animals↗
PubMed2004

Integrated control of lung fluid balance.

This review summarizes the highlights of the EB2004 symposium that dealt with the integrated aspects of the lung fluid balance. It is apparent that maintenance of lung fluid balance requires the proper functioning of vascular endothelial and alveolar epithelial barriers. Under physiological conditions, the transcytotic pathway requiring repeated fission-fusion events of the caveolar membrane with other caveolae solely transports albumin. Caveolin-1, which forms caveolae, and albumin-binding proteins play a central role in signaling the transcytosis of albumin. Signals responsible for increasing endothelial permeability in lung microvessels in response to inflammatory mediators were also described. These studies in gene knockout mouse models revealed the importance of Ca(2+) signaling via store-operated transient receptor channel 4 and the activation of endothelial myosin light chain kinase isoform in mediating the increase in microvessel permeability. Increases in the cytosolic Ca(2+) in situ in microvessel endothelia can occur by mitochondria-dependent as well as mitochondria-independent pathways (such as the endoplasmic reticulum). Both these pathways, by triggering endothelial cell activation, may result in lung microvascular injury. The resolution of alveolar edema, requiring clearance of fluid from the air space, is another area of intense investigation in animal models. Although beta-adrenergic agonists can activate alveolar fluid clearance, signaling pathways regulating these events in intact alveoli remain to be established. Development of mouse models in which the function of regulatory proteins (identified in cell culture studies) can be systematically analyzed will provide a better and more integrated picture of lung fluid balance. In vivo veritas!

Animals↗
PubMed2003

Future research directions in acute lung injury: summary of a National Heart, Lung, and Blood Institute working group.

Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), are syndromes of acute respiratory failure that result from acute pulmonary edema and inflammation. The development of ALI/ARDS is associated with several clinical disorders including direct pulmonary injury from pneumonia and aspiration as well as indirect pulmonary injury from trauma, sepsis, and other disorders such as acute pancreatitis and drug overdose. Although mortality from ALI/ARDS has decreased in the last decade, it remains high. Despite two major advances in treatment, low VT ventilation for ALI/ARDS and activated protein C for severe sepsis (the leading cause of ALI/ARDS), additional research is needed to develop specific treatments and improve understanding of the pathogenesis of these syndromes. The NHLBI convened a working group to develop specific recommendations for future ALI/ARDS research. Improved understanding of disease heterogeneity through use of evolving biologic, genomic, and genetic approaches should provide major new insights into pathogenesis of ALI. Cellular and molecular methods combined with animal and clinical studies should lead to further progress in the detection and treatment of this complex disease.

Academies and Institutes↗
PubMed2003

Endothelial barrier strengthening by activation of focal adhesion kinase.

Endothelial cell barrier (EC) properties regulate blood tissue fluid flux. To determine the role of endothelial-matrix interactions in barrier regulation, we induced cell shrinkage by exposing confluent endothelial monolayers to hyperosmolarity. The dominant effect of a 15-min hyperosmolar exposure was an increase in the trans-endothelial electrical resistance, indicating the induction of barrier strengthening. Hyperosmolar exposure also increased activity of focal adhesion kinase and E-cadherin accumulation at the cell periphery. Concomitantly, the density of actin filaments increased markedly. In EC monolayers stably expressing constitutively active or dominant negative isoforms of Rac1, the actin response to hyperosmolar exposure was enhanced or blocked, respectively, although the response in trans-endothelial resistance was unaffected, indicating that the endothelial barrier enhancement occurred independently of actin. However, in monolayers expressing a kinase-deficient mutant of focal adhesion kinase, the hyperosmolarity-induced increases in activity of focal adhesion and peripheral E-cadherin enhancement were blocked and the induced increase of electrical resistance was markedly blunted. These findings indicate that in EC exposed to hyperosmolar challenge, the involvement of focal adhesion kinase was critical in establishing barrier strengthening.

Animals↗
PubMed2003

High tidal volume ventilation induces proinflammatory signaling in rat lung endothelium.

Alveolar overdistension during mechanical ventilation causes leukocyte sequestration, leading to lung injury. However, underlying endothelial cell (EC) mechanisms are undefined. In a new approach, we exposed isolated blood-perfused rat lungs to high tidal volume ventilation (HV) for 2 h, then obtained fresh lung endothelial cells (FLEC) by immunosorting at 4 degrees C. Immunoblotting experiments indicated that as compared with FLEC derived from lungs ventilated at low volume (LV), HV markedly enhanced tyrosine phosphorylation (TyrP). The tyrosine kinase blocker, genistein, inhibited this response. HV also induced focal adhesion (FA) formation in FLEC, as detected by immunofluorescent aggregates of the alpha(v)beta(3) integrin that co-localized with aggregations of focal adhesion kinase (FAK). Immunoprecipitation and blotting experiments revealed that HV increased TyrP of the FA protein, paxillin. In addition, HV induced a paxillin-associated P-selectin expression on FLEC that was also inhibited by genistein. However, HV did not increase lung water. These results indicate that in HV, EC signaling in situ causes FA formation and induces TyrP-dependent P-selectin expression. These signaling mechanisms may promote leukocyte-mediated responses in HV.

Animals↗
PubMed2003

Mechano-oxidative coupling by mitochondria induces proinflammatory responses in lung venular capillaries.

Elevation of lung capillary pressure causes exocytosis of the leukocyte adhesion receptor P-selectin in endothelial cells (ECs), indicating that lung ECs generate a proinflammatory response to pressure-induced stress. To define underlying mechanisms, we followed the EC signaling sequence leading to P-selectin exocytosis through application of real-time, in situ fluorescence microscopy in lung capillaries. Pressure elevation increased the amplitude of cytosolic Ca(2+) oscillations that triggered increases in the amplitude of mitochondrial Ca(2+) oscillations and in reactive oxygen species (ROS) production. Responses to blockers of the Ca(2+) oscillations and of mitochondrial electron transport indicated that the ROS production was Ca(2+) dependent and of mitochondrial origin. A new proinflammatory mechanism was revealed in that pressure-induced exocytosis of P-selectin was inhibited by both antioxidants and mitochondrial inhibitors, indicating that the exocytosis was driven by mitochondrial ROS. In this signaling pathway mitochondria coupled pressure-induced Ca(2+) oscillations to the production of ROS that in turn acted as diffusible messengers to activate P-selectin exocytosis. These findings implicate mitochondrial mechanisms in the lung's proinflammatory response to pressure elevation and identify mitochondrial ROS as critical to P-selectin exocytosis in lung capillary ECs.

Animals↗