Timothy M. Moore

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+and permeability

Cytosolic Ca2+ concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+ induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 μM) produced higher [Ca2+]ilevels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]iresponse in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+increased RPMVEC [Ca2+]ito a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]iin RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 μM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]ipromotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]isignaling pathways or dominant Ca2+-independent mechanisms from controlling cellular gap formation and permeability.Cytosolic Ca2+ concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+ induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 microM) produced higher [Ca2+]i levels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]i response in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+ increased RPMVEC [Ca2+]i to a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]i in RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 microM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]i promotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]i signaling pathways or dominant Ca(2+)-independent mechanisms from controlling cellular gap formation and permeability.

Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway

Previous work from our laboratory has demonstrated that infection with influenza A/Bangkok/1/79 (H3N2), a relatively mild strain of the virus, caused much more severe pneumonitis in selenium (Se)‐deficient mice than in Se‐adequate mice. Here we report that the increased virulence observed in the Se‐deficient mice is due to mutations in the influenza virus genome, resulting in a more virulent genotype. Most of the mutations occurred in the gene for the M1 matrix protein, an internal protein that is thought to be relatively stable. A total of 29 nucleotide changes were observed in this gene, and all 29 changes were identical in three separate isolates taken from three different Se‐deficient mice. In contrast, only one to three mutations were seen in the genes for the hemagglutinin or neuraminidase proteins, surface antigens that are known to be highly variable. Once the mutations have occurred, even hosts with normal nutritional status are susceptible to the newly virulent strain. This work, in conjunction with our earlier work with coxsackievirus, shows that specific nutritional deficiencies can have a profound impact on the genome of RNA viruses. Poor nutritional status in the host may contribute to the emergence of new viral strains.

Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP.

Pulmonary endothelium forms a semiselective barrier that regulates fluid balance and leukocyte trafficking. During the course of lung inflammation, neurohumoral mediators and oxidants act on endothelial cells to induce intercellular gaps permissive for transudation of proteinaceous fluid from blood into the interstitium. Intracellular signals activated by neurohumoral mediators and oxidants that evoke intercellular gap formation are incompletely understood. Cytosolic Ca2+ concentration ([Ca2+]i) and cAMP are two signals that importantly dictate cell-cell apposition. Although increased [Ca2+]ipromotes disruption of the macrovascular endothelial cell barrier, increased cAMP enhances endothelial barrier function. Furthermore, during the course of inflammation, elevated endothelial cell [Ca2+]idecreases cAMP to facilitate intercellular gap formation. Given the significance of both [Ca2+]iand cAMP in mediating cell-cell apposition, this review addresses potential sites of cross talk between these two intracellular signaling pathways. Emerging data also indicate that endothelial cells derived from different vascular sites within the pulmonary circulation exhibit distinct sensitivities to permeability-inducing stimuli; that is, elevated [Ca2+]ipromotes macrovascular but not microvascular barrier disruption. Thus this review also considers the roles of [Ca2+]iand cAMP in mediating site-specific alterations in endothelial permeability.Pulmonary endothelium forms a semiselective barrier that regulates fluid balance and leukocyte trafficking. During the course of lung inflammation, neurohumoral mediators and oxidants act on endothelial cells to induce intercellular gaps permissive for transudation of proteinaceous fluid from blood into the interstitium. Intracellular signals activated by neurohumoral mediators and oxidants that evoke intercellular gap formation are incompletely understood. Cytosolic Ca2+ concentration ([Ca2+]i) and cAMP are two signals that importantly dictate cell-cell apposition. Although increased [Ca2+]i promotes disruption of the macrovascular endothelial cell barrier, increased cAMP enhances endothelial barrier function. Furthermore, during the course of inflammation, elevated endothelial cell [Ca2+]i decreases cAMP to facilitate intercellular gap formation. Given the significance of both [Ca2+]i and cAMP in mediating cell-cell apposition, this review addresses potential sites of cross talk between these two intracellular signaling pathways. Emerging data also indicate that endothelial cells derived from different vascular sites within the pulmonary circulation exhibit distinct sensitivities to permeability-inducing stimuli; that is, elevated [Ca2+]i promotes macrovascular but not microvascular barrier disruption. Thus this review also considers the roles of [Ca2+]i and cAMP in mediating site-specific alterations in endothelial permeability.

Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1

Activation of Ca2+ entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells. The mode of Ca2+ entry regulating cell shape is unknown. We hypothesized that activation of store-operated Ca2+ channels (SOCs) is sufficient to promote cell shape change necessary for these processes. SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca2+ that was dependent on a membrane current having a net inward component of 5.45 +/- 0.90 pA/pF at -80 mV. Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca2+ entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin). Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential (trp) gene have been proposed to form Ca2+ entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells. Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6. Our study indicates that 1) Ca2+ entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs.Activation of Ca2+ entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells. The mode of Ca2+ entry regulating cell shape is unknown. We hypothesized that activation of store-operated Ca2+ channels (SOCs) is sufficient to promote cell shape change necessary for these processes. SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca2+ that was dependent on a membrane current having a net inward component of 5.45 ± 0.90 pA/pF at -80 mV. Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca2+ entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin). Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential ( trp) gene have been proposed to form Ca2+ entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells. Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6. Our study indicates that 1) Ca2+ entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs.

Dominant regulation of interendothelial cell gap formation by calcium-inhibited type 6 adenylyl cyclase

Acute transitions in cytosolic calcium ([Ca2+]i) through store-operated calcium entry channels catalyze interendothelial cell gap formation that increases permeability. However, the rise in [Ca2+]i only disrupts barrier function in the absence of a rise in cAMP. Discovery that type 6 adenylyl cyclase (AC6; EC 4.6.6.1) is inhibited by calcium entry through store-operated calcium entry pathways provided a plausible explanation for how inflammatory [Ca2+]i mediators may decrease cAMP necessary for endothelial cell gap formation. [Ca2+]i mediators only modestly decrease global cAMP concentrations and thus, to date, the physiological role of AC6 is unresolved. Present studies used an adenoviral construct that expresses the calcium-stimulated AC8 to convert normal calcium inhibition into stimulation of cAMP, within physiologically relevant concentration ranges. Thrombin stimulated a dose-dependent [Ca2+]i rise in both pulmonary artery (PAECs) and microvascular (PMVEC) endothelial cells, and promoted intercellular gap formation in both cell types. In PAECs, gap formation was progressive over 2 h, whereas in PMVECs, gap formation was rapid (within 10 min) and gaps resealed within 2 h. Expression of AC8 resulted in a modest calcium stimulation of cAMP, which virtually abolished thrombin-induced gap formation in PMVECs. Findings provide the first direct evidence that calcium inhibition of AC6 is essential for endothelial gap formation.

Segmental regulation of pulmonary vascular permeability by store-operated Ca2+ entry

An intact endothelial cell barrier maintains normal gas exchange in the lung, and inflammatory conditions result in barrier disruption that produces life-threatening hypoxemia. Activation of store-operated Ca2+ (SOC) entry increases the capillary filtration coefficient ( K f,c) in the isolated rat lung; however, activation of SOC entry does not promote permeability in cultured rat pulmonary microvascular endothelial cells. Therefore, current studies tested whether activation of SOC entry increases macro- and/or microvascular permeability in the intact rat lung circulation. Activation of SOC entry by the administration of thapsigargin induced perivascular edema in pre- and postcapillary vessels, with apparent sparing of the microcirculation as evaluated by light microscopy. Scanning and transmission electron microscopy revealed that the leak was due to gaps in vessels ≥ 100 μm, consistent with the idea that activation of SOC entry influences macrovascular but not microvascular endothelial cell shape. In contrast, ischemia and reperfusion induced microvascular endothelial cell disruption independent of Ca2+ entry, which similarly increased K f,c. These data suggest that 1) activation of SOC entry is sufficient to promote macrovascular barrier disruption and 2) unique mechanisms regulate pulmonary micro- and macrovascular endothelial barrier functions.

Microtubule Motors Regulate ISOC Activation Necessary to Increase Endothelial Cell Permeability

Calcium store depletion activates multiple ion channels, including calcium-selective and nonselective channels. Endothelial cells express TRPC1 and TRPC4 proteins that contribute to a calcium-selective store-operated current, ISOC. Whereas thapsigargin activates the ISOC in pulmonary artery endothelial cells (PAECs), it does not activate ISOC in pulmonary microvascular endothelial cells (PMVECs), despite inducing a significant rise in global cytosolic calcium. Endoplasmic reticulum exhibits retrograde distribution in PMVECs when compared with PAECs. We therefore sought to determine whether endoplasmic reticulum-to-plasma membrane coupling represents an important determinant of ISOC activation in PAECs and PMVECs. Endoplasmic reticulum organization is controlled by microtubules, because nocodozole induced microtubule disassembly and caused retrograde endoplasmic reticulum collapse in PMVECs. In PMVECs, rolipram treatment produced anterograde endoplasmic reticulum distribution and revealed a thapsigargin-activated ISOC that was abolished by nocodozole and taxol. Microtubule motors control organelle distribution along microtubule tracks, with the dynein motor causing retrograde movement and the kinesin motor causing anterograde movement. Dynamitin expression reduces dynein motor function inducing anterograde endoplasmic reticulum transport, which allows for direct activation of ISOC by thapsigargin in PMVECs. In contrast, expression of dominant negative kinesin light chain reduces kinesin motor function and induces retrograde endoplasmic reticulum transport; dominant negative kinesin light chain expression prevented the direct activation of ISOC by thapsigargin in PAECs. ISOC activation is an important step leading to disruption of cell-cell adhesion and increased macromolecular permeability. Thus, microtubule motor function plays an essential role in activating cytosolic calcium transitions through the membrane ISOC channel leading to endothelial barrier disruption.

Segmental hemodynamics during partial liquid ventilation in isolated rat lungs.

UNLABELLED Partial liquid ventilation (PLV) is a means of ventilatory support in which gas ventilation is carried out in a lung partially filled with a perfluorocarbon liquid capable of supporting gas exchange. Recently, this technique has been proposed as an adjunctive therapy for cardiac arrest, during which PLV with cold perfluorocarbons might rapidly cool the intrathoracic contents and promote cerebral protective hypothermia while not interfering with gas exchange. A concern during such therapy will be the effect of PLV on pulmonary hemodynamics during very low blood flow conditions. In the current study, segmental (i.e. precapillary, capillary, and postcapillary) hemodynamics were studied in the rat lung using a standard isolated lung perfusion system at a flow rate of 6 ml/min ( approximately 5% normal cardiac output). Lungs received either gas ventilation or 5 or 10 ml/kg PLV. Segmental pressures and vascular resistances were determined, as was transcapillary fluid flux. The relationship between individual hemodynamic parameters and PLV dose was examined using linear regression, with n=5 in each study group. PLV at both the 5 and 10 ml/kg dose produced no detectable changes in pulmonary blood flow or in transcapillary fluid flux (all R(2) values<0.20). CONCLUSION In an isolated perfused lung model of low flow conditions, normal segmental hemodynamic behavior was preserved during liquid ventilation. These data support further investigation of this technique as an adjunct to cardiopulmonary resuscitation.

Evaluation of endothelial barrier damage in lungs

It is well known that sepsis, periods of lung ischemia followed by reperfusion, various ventilation regimens, elevation of cerebral spinal fluid pressure, and ischemia/ reperfusion of the small bowel cause extensive pulmonary edema [1–4]. However, the mechanisms associated with producing edema under these conditions are difficult to ascertain when studying the lung in situ, since both elevations in microvascular pressure and damage to the endothelial barrier act synergistically to produce pulmonary edema. Unfortunately, the measures needed to determine the biophysical permeability characteristics of the endothelial barrier in the lung require either measurement of lung weight changes with known increments in microvascular pressure, evaluation of lung lymph flow and its protein content, or the protein content of the lung’s extravascular tissue. The latter two of these measures are difficult to obtain for in situ rat lung preparations, but have been used successfully in both chronic and acute sheep and dog lung models where lymph flow and vascular pressures can be obtained [5–7]. Over the last ten years, isolated rat lung models have been used to study various lung pathologies, such as the inflammatory response associated with ischemia and reperfusion. Using these experimental models, numerous mechanisms have been identified that modulate the selectivity of the endothelial barrier [3,4]. However, it is also important to understand how the lung endothelial barrier behaves in situ in response to injury. This information is necessary in order to determine how various clinical interventions and drugs can prevent or reverse the damage. Though isolated organs are important models to identify mechanisms associated with lung injury, there is no guarantee that the same mechanisms identified in the ex vivo lung apply when the lung is in situ and affected by numerous substances released from tissues other than lung. In this review, we will present the basic biophysical parameters that define the transvascular flux of proteins and solvent in the lung. These measures will be discussed relative to their applicability to in situ lung studies.