Abstract-Although the formation of hydrostatic lung edema is generally attributed to imbalanced Starling forces, recent data show that lung endothelial cells respond to increased vascular pressure and may thus regulate vascular permeability and edema formation. In combining real-time optical imaging of the endothelial Ca 2ϩ concentration ([Ca 2ϩ ] i ) and NO production with filtration coefficient (K f ) measurements in the isolated perfused lung, we identified a series of endothelial responses that constitute a negative-feedback loop to protect the microvascular barrier. Elevation of lung microvascular pressure was shown to increase endothelial [Ca 2ϩ ] i via activation of transient receptor potential vanilloid 4 (TRPV4) channels. The endothelial [Ca 2ϩ ] i transient increased K f via activation of myosin light-chain kinase and simultaneously stimulated NO synthesis. In TRPV4 deficient mice, pressure-induced increases in endothelial [Ca 2ϩ ] i , NO synthesis, and lung wet/dry weight ratio were largely blocked. Endothelial NO formation limited the permeability increase by a cGMP-dependent attenuation of the pressure-induced [Ca 2ϩ ] i response. Inactivation of TRPV4 channels by cGMP was confirmed by whole-cell patch-clamp of pulmonary microvascular endothelial cells and intravital imaging of endothelial [Ca 2ϩ ] i . Hence, pressure-induced endothelial Ca 2ϩ influx via TRPV4 channels increases lung vascular permeability yet concomitantly activates an NO-mediated negative-feedback loop that protects the vascular barrier by a cGMP-dependent attenuation of the endothelial [Ca 2ϩ ] i response. The identification of this novel regulatory pathway gives rise to new treatment strategies, as demonstrated in vivo in rats with acute myocardial infarction in which inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil reduced hydrostatic lung edema. Key Words: pulmonary edema Ⅲ vascular permeability Ⅲ vascular endothelium Ⅲ phosphodiesterase type 5 inhibitor Ⅲ nitric oxide T he pathogenesis of hydrostatic lung edema has been attributed predominantly to an imbalance in Starling forces, ie, fluid extravasation attributable to an increased hydrostatic or reduced oncotic pressure gradient across the microvascular barrier. This classic view has been challenged by the findings of Parker and Ivey in isolated perfused rat lungs, which demonstrated an increase in lung filtration coefficient (K f ) following elevation of left atrial pressure (P LA ). 1 This increase was attenuated by the ␤-adrenergic agonist isoproterenol, indicating that the K f increase was not only caused by an enlarged vascular surface area but also resulted from an increase in vascular permeability that could be counteracted via the cAMP signaling pathway. The latter finding suggests that active endothelial responses may contribute critically to the formation of hydrostatic lung edema.By use of real-time fluorescence imaging techniques, we recently identified such endothelial responses to an acute elevation in hydrostatic pres...
Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wildtype mice and in lungs from mice lacking Cx40 (Cx40 -/-). In vivo, hypoxemia was more severe in Cx40 -/-mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not Cx40 -/-, mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α 1G subtype Ca 2+ channels, cytosolic phospholipase A 2 , and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner. IntroductionHypoxic pulmonary vasoconstriction (HPV) is a fundamental physiological mechanism by which the lung optimizes ventilation/ perfusion (V/Q) matching, redirecting blood flow from poorly to better ventilated areas (1). Yet in cases of global hypoxia, HPV may unfavorably increase total pulmonary vascular resistance and right ventricular afterload, thus contributing to the clinical pathology of pulmonary hypertension and cor pulmonale in chronic hypoxic lung diseases or to pulmonary edema at high altitude (1, 2). While the relevance of HPV has been recognized for over 60 years, the underlying oxygen sensing and signal transduction processes remain a topic of intense research and controversy. Current concepts of HPV are largely based on the notion that pulmonary arterial smooth muscle cells (PASMCs) constitute both the sensor and the transducer of the hypoxic signal as well as its contractile effector (1), while the role of the vascular endothelium is at best considered that of a modulating bystander.From a conceptual standpoint, the ideal site for an oxygen sensor in HPV is within the actual area of pulmonary gas exchange,
BackgroundMast cells (MCs) are implicated in inflammation and tissue remodeling. Accumulation of lung MCs is described in pulmonary hypertension (PH); however, whether MC degranulation and c-kit, a tyrosine kinase receptor critically involved in MC biology, contribute to the pathogenesis and progression of PH has not been fully explored.MethodsPulmonary MCs of idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline-injected rats (MCT-rats) were examined by histochemistry and morphometry. Effects of the specific c-kit inhibitor PLX and MC stabilizer cromolyn sodium salt (CSS) were investigated in MCT-rats both by the preventive and therapeutic approaches. Hemodynamic and right ventricular hypertrophy measurements, pulmonary vascular morphometry and analysis of pulmonary MC localization/counts/activation were performed in animal model studies.ResultsThere was a prevalence of pulmonary MCs in IPAH patients and MCT-rats as compared to the donors and healthy rats, respectively. Notably, the perivascular MCs were increased and a majority of them were degranulated in lungs of IPAH patients and MCT-rats (p < 0.05 versus donor and control, respectively). In MCT-rats, the pharmacological inhibitions of MC degranulation and c-kit with CSS and PLX, respectively by a preventive approach (treatment from day 1 to 21 of MCT-injection) significantly attenuated right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Moreover, vascular remodeling, as evident from the significantly decreased muscularization and medial wall thickness of distal pulmonary vessels, was improved. However, treatments with CSS and PLX by a therapeutic approach (from day 21 to 35 of MCT-injection) neither improved hemodynamics and RVH nor vascular remodeling.ConclusionsThe accumulation and activation of perivascular MCs in the lungs are the histopathological features present in clinical (IPAH patients) and experimental (MCT-rats) PH. Moreover, the accumulation and activation of MCs in the lungs contribute to the development of PH in MCT-rats. Our findings reveal an important pathophysiological insight into the role of MCs in the pathogenesis of PH in MCT- rats.
The monoamines octopamine (OA) and tyramine (TA) modulate numerous behaviours and physiological processes in invertebrates. Nevertheless, it is not clear whether these invertebrate counterparts of norepinephrine are important regulators of metabolic and life history traits. We show that flies (Drosophila melanogaster) lacking OA are more resistant to starvation, while their overall life span is substantially reduced compared with control flies. In addition, these animals have increased body fat deposits, reduced physical activity and a reduced metabolic resting rate. Increasing the release of OA from internal stores induced the opposite effects. Flies devoid of both OA and TA had normal body fat and metabolic rates, suggesting that OA and TA act antagonistically. Moreover, OA-deficient flies show increased insulin release rates. We inferred that the OA-mediated control of insulin release accounts for a substantial proportion of the alterations observed in these flies. Apparently, OA levels control the balance between thrifty and expenditure metabolic modes. Thus, changes in OA levels in response to external and internal signals orchestrate behaviour and metabolic processes to meet physiological needs. Moreover, chronic deregulation of the corresponding signalling systems in humans may be associated with metabolic disorders, such as obesity or diabetes.
Despite clinical and histologic vascular remodeling in all patients with PH-COPD and PH-IPF, differential gene expression pattern was present in pulmonary artery profiles. Several genes involved in retinol metabolism and ECM receptor interaction enable discrimination of vascular remodeling in PH-IPF or PH-COPD. This suggests that pulmonary arterial remodeling in PH-COPD and PH-IPF is caused by different molecular mechanisms and may require specific therapeutic options.
In the formation of COPI vesicles, interactions take place between the coat protein coatomer and membrane proteins: either cargo proteins for retrieval to the endoplasmic reticulum (ER) or proteins that cycle between the ER and the Golgi. While the binding sites on coatomer for ER residents have been characterized, how cycling proteins bind to the COPI coat is still not clear. In order to understand at a molecular level the mechanism of uptake of such proteins, we have investigated the binding to coatomer of p24 proteins as examples of cycling proteins as well as that of ER-resident cargos. The p24 proteins required dimerization to interact with coatomer at two independent binding sites in ␥-COP. In contrast, ER-resident cargos bind to coatomer as monomers and to sites other than ␥-COP. The COPI coat therefore discriminates between p24 proteins and ER-resident proteins by differential binding involving distinct subunits.COPI vesicles mediate transport in the early secretory pathway and are involved in the retrieval of endoplasmic reticulum (ER)-resident proteins from the Golgi apparatus to the ER (23). Numerous transmembrane ER-resident proteins have conserved KKXX motifs at the extreme C termini of their cytoplasmic tails (17). As there is an ongoing flow of transport from the ER to the Golgi, ER residents may escape the organelle and thus need to be retrieved to their proper location. The KKXX motifs are recognized by the coatomer complex (4), which coats COPI vesicles (6,38,42). Therefore, escaped ER residents can be retrieved from the Golgi through the COPI system by direct interaction between a conserved recognition sequence and the coat protein (24). In contrast to escaped ER residents, which are vectorially transported to their proper localization, other transmembrane proteins are found in the early secretory pathway, where they constitutively cycle between the ER and the Golgi by using the COPII and the COPI systems, such as members of the p24 family of type I transmembrane proteins, e.g. (29). The p24 proteins were shown to be major components of COPI-coated vesicles (39,41). Their cytoplasmic tails have two conserved motifs: a diphenylalanine motif and a dibasic motif. While in mammalian p25 and its yeast orthologs the dibasic motif is identical to the KKXX motif, this is not the case for all the other family members. Yeast and mammalian p25 proteins were described as depending on their KKXX sequence in order to bind to an ␣/␤Ј/ε-COP subcomplex of coatomer, whereas yeast and mammalian p24 and yeast p26 were described as depending on their diphenylalanine motif in order to bind to a ␤/␥/-COP subcomplex (10). Finally, yeast p23 and mammalian p26 were described as not binding to coatomer (10). However, this study is hard to interpret, since cytoplasmic tails of yeast or mammalian p24 proteins were both used with mammalian coatomer. Moreover, other studies presented contradictory results showing that p23 and p25 are the only p24 proteins that can bind to coatomer (5), while yet others described binding of ...
Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of pulmonary hypertension. Here, we have undertaken a compartment-specific study to elucidate the expression profile of collagens and their processing enzymes in donor and idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries. Predominant intimal, but also medial and perivascular, remodeling and reduced lumen diameter were detected in IPAH pulmonary arteries. Two-photon microscopy demonstrated accumulation of collagen fibers. Quantification of collagen in pulmonary arteries revealed collagen accumulation mainly in the intima of IPAH pulmonary arteries compared with donors. Laser capture-microdissected pulmonary artery profiles (intima+media and perivascular tissue) were analyzed by real-time PCR for ECM gene expression. In the intima+media of IPAH vessels, collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) 33 were higher expressed, whereas MMP10, ADAM17, TIMP1, and TIMP3 were less abundant. Localization of COLXVIII, its cleavage product endostatin, and MMP10, ADAM33, and TIMP1 was confirmed in pulmonary arteries by immunohistochemistry. ELISA for collagen XVIII/endostatin demonstrated significantly elevated plasma levels in IPAH patients compared with donors, whereas circulating MMP10, ADAM33, and TIMP1 levels were similar between the two groups. Endostatin levels were correlated with pulmonary arterial wedge pressure, and established prognostic markers of IPAH, right atrial pressure, cardiac index, 6-min walking distance, NH2-terminal pro-brain natriuretic peptide, and uric acid. Expression of unstudied collagens, MMPs, ADAMs, and TIMPs were found to be significantly altered in IPAH intima+media. Elevated levels of circulating collagen XVIII/endostatin are associated with markers of a poor prognosis.
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