High-intensity interval training, but not continuous training, reverses right ventricular hypertrophy and dysfunction in a rat model of pulmonary hypertension.

Exercise is beneficial in pulmonary arterial hypertension (PAH), although studies to date indicate little effect on the elevated pulmonary pressures or maladaptive right ventricle (RV) hypertrophy associated with the disease. For chronic left ventricle failure, high-intensity interval training (HIIT) promotes greater endothelial stimulation and superior benefit than customary continuous exercise training (CExT); however, HIIT has not been tested for PAH. Therefore, here we investigated acute and chronic responses to HIIT vs. CExT in a rat model of monocrotaline (MCT)-induced mild PAH. Six weeks of treadmill training (5 times/wk) were performed, as either 30 min HIIT or 60 min low-intensity CExT. To characterize acute hemodynamic responses to the two approaches, novel recordings of simultaneous pulmonary and systemic pressures during running were obtained at pre- and 2, 4, 6, and 8 wk post-MCT using long-term implantable telemetry. MCT-induced decrement in maximal aerobic capacity was ameliorated by both HIIT and CExT, with less pronounced pulmonary vascular remodeling and no increase in RV inflammation or apoptosis observed. Most importantly, only HIIT lowered RV systolic pressure, RV hypertrophy, and total pulmonary resistance, and prompted higher cardiac index that was complemented by a RV increase in the positive inotrope apelin and reduced fibrosis. HIIT prompted a markedly pulsatile pulmonary pressure during running and was associated with greater lung endothelial nitric oxide synthase after 6 wk. We conclude that HIIT may be superior to CExT for improving hemodynamics and maladaptive RV hypertrophy in PAH. HIIT's superior outcomes may be explained by more favorable pulmonary vascular endothelial adaptation to the pulsatile HIIT stimulus.

Novel assessment of haemodynamic kinetics with acute exercise in a rat model of pulmonary arterial hypertension.

NEW FINDINGS: What is the central question of this study? The acute effect of exercise at moderately high intensity on already-elevated pulmonary arterial pressures and right ventricular wall stress in a rat model of pulmonary arterial hypertension (PAH) is unknown. What is the main finding and its importance? We show, for the first time, that in a rat model of PAH, exercise induces an acute reduction in pulmonary artery pressure associated with lung endothelial nitric oxide synthase activation, without evidence of acute right ventricular inflammation or myocyte apoptosis. Haemodynamic measures obtained with traditional invasive methodology as well as novel implantable telemetry reveal an exercise-induced 'window' of pulmonary hypertension alleviation, supporting future investigations of individualized exercise as therapy in PAH. Exercise improves outcomes of multiple chronic conditions, but controversial results, including increased pulmonary artery (PA) pressure, have prevented its routine implementation in pulmonary arterial hypertension (PAH), an incurable disease that drastically reduces exercise tolerance. Individualized, optimized exercise prescription for PAH requires a better understanding of disease-specific exercise responses. We investigated the acute impact of exercise on already-elevated PA pressure and right ventricular (RV) wall stress and inflammation in a rat model of PAH (PAH group, n = 12) induced once by monocrotaline (50 mg kg(-1) , i.p.; 2 weeks), compared with healthy control animals (n = 8). Single bouts of exercise consisted of a 45 min treadmill run at 75% of individually determined aerobic capacity (V̇O2max). Immediately after exercise, measurements of RV systolic pressure and systemic pressure were made via jugular and carotid cannulation, and were followed by tissue collection. Monocrotaline induced moderate PAH, evidenced by RV hypertrophy, decreased V̇O2max, PA muscularization, and RV and skeletal muscle cytoplasmic glycolysis detected by increased expression of glucose transporter-1. Acute exercise normalized the monocrotaline-induced elevation in RV systolic pressure and augmented pulmonary endothelial nitric oxide synthase activation, without evidence of increased RV inflammation or apoptosis. Real-time recordings of pulmonary and systemic pressures during and after single bouts of exercise made using novel implantable telemetry in the same animal for up to 11 weeks after monocrotaline (40 mg kg(-1) ) corroborated the finding of acute PA pressure decreases with exercise in PAH. The PA pressure-lowering effects of individualized exercise associated with RV-neutral effects and increases in vasorelaxor signalling encourage further development of optimized exercise regimens as adjunctive PAH therapy.

RTP801 is required for ceramide-induced cell-specific death in the murine lung.

Key host responses to the stress induced by environmental exposure to cigarette smoke (CS) are responsible for initiating pathogenic effects that may culminate in emphysema development. CS increases lung ceramides, sphingolipids involved in oxidative stress, structural alveolar cell apoptosis, and inhibition of apoptotic cell clearance by alveolar macrophages, leading to the development of emphysema-like pathology. RTP801, a hypoxia and oxidative stress sensor, is also increased by CS, and has been recently implicated in both apoptosis and inflammation. We investigated whether inductions of ceramide and RTP801 are mechanistically linked, and evaluated their relative importance in lung cell apoptosis and airspace enlargement in vivo. As reported, direct lung instillation of either RTP801 expression plasmid or ceramides in mice triggered alveolar cell apoptosis and oxidative stress. RTP801 overexpression up-regulated lung ceramide levels 2.6-fold. In turn, instillation of lung ceramides doubled the lung content of RTP801. Cell sorting after lung tissue dissociation into single-cell suspension showed that ceramide triggers both endothelial and epithelial cell apoptosis in vivo. Interestingly, mice lacking rtp801 were protected against ceramide-induced apoptosis of epithelial type II cells, but not type I or endothelial cells. Furthermore, rtp801-null mice were protected from ceramide-induced alveolar enlargement, and exhibited improved static lung compliance compared with wild-type mice. In conclusion, ceramide and RTP801 participate in alveolar cell apoptosis through a process of mutual up-regulation, which may result in self-amplification loops, leading to alveolar damage.

17ß-Estradiol attenuates hypoxic pulmonary hypertension via estrogen receptor-mediated effects.

RATIONALE: 17ß-Estradiol (E2) attenuates hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension (HPH) through an unknown mechanism that may involve estrogen receptors (ER) or E2 conversion to catecholestradiols and methoxyestradiols with previously unrecognized effects on cardiopulmonary vascular remodeling. OBJECTIVES: To determine the mechanism by which E2 exerts protective effects in HPH. METHODS: Male rats were exposed to hypobaric hypoxia while treated with E2 (75 µg/kg/d) or vehicle. Subgroups were cotreated with pharmacologic ER-antagonist or with inhibitors of E2-metabolite conversion. Complementary studies were performed in rats cotreated with selective ERα- or ERß-antagonist. Hemodynamic and pulmonary artery (PA) and right ventricular (RV) remodeling parameters, including cell proliferation, cell cycle, and autophagy, were measured in vivo and in cultured primary rat PA endothelial cells. MEASUREMENTS AND MAIN RESULTS: E2 significantly attenuated HPH endpoints. Hypoxia increased ERß but not ERα lung vascular expression. Co-treatment with nonselective ER inhibitor or ERα-specific antagonist rendered hypoxic animals resistant to the beneficial effects of E2 on cardiopulmonary hemodynamics, whereas ERα- and ERß-specific antagonists opposed the remodeling effects of E2. In contrast, inhibition of E2-metabolite conversion did not abolish E2 protection. E2-treated hypoxic animals exhibited reduced ERK1/2 activation and increased expression of cell-cycle inhibitor p27(Kip1) in lungs and RV, with up-regulation of lung autophagy. E2-induced signaling was recapitulated in hypoxic but not normoxic endothelial cells, and was associated with decreased vascular endothelial growth factor secretion and cell proliferation. CONCLUSIONS: E2 attenuates hemodynamic and remodeling parameters in HPH in an ER-dependent manner, through direct antiproliferative mechanisms on vascular cells, which may provide novel nonhormonal therapeutic targets for HPH.

Effect of cigarette smoke exposure and structural modifications on the α-1 Antitrypsin interaction with caspases.

α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.

Two-photon imaging within the murine thorax without respiratory and cardiac motion artifact.

Intravital microscopy has been recognized for its ability to make physiological measurements at cellular and subcellular levels while maintaining the complex natural microenvironment. Two-photon microscopy (TPM), using longer wavelengths than single-photon excitation, has extended intravital imaging deeper into tissues, with minimal phototoxicity. However, due to a relatively slow acquisition rate, TPM is especially sensitive to motion artifact, which presents a challenge when imaging tissues subject to respiratory and cardiac movement. Thoracoabdominal organs that cannot be exteriorized or immobilized during TPM have generally required the use of isolated, pump-perfused preparations. However, this approach entails significant alteration of normal physiology, such as a lack of neural inputs, increased vascular resistance, and leukocyte activation. We adapted techniques of intravital microscopy that permitted TPM of organs maintained within the thoracoabdominal cavity of living, breathing rats or mice. We obtained extended intravital TPM imaging of the intact lung, arguably the organ most susceptible to both respiratory and cardiac motion. Intravital TPM detected the development of lung microvascular endothelial activation manifested as increased leukocyte adhesion and plasma extravasation in response to oxidative stress inducers PMA or soluble cigarette smoke extract. The pulmonary microvasculature and alveoli in the intact animal were imaged with comparable detail and fidelity to those in pump-perfused animals, opening the possibility for TPM of other thoracoabdominal organs under physiological and pathophysiological conditions.

Adipose stem cell treatment in mice attenuates lung and systemic injury induced by cigarette smoking.

RATIONALE: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery. OBJECTIVES: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema. METHODS: Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors. MEASUREMENTS AND MAIN RESULTS: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro. CONCLUSIONS: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.

Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides.

The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.

Anti-type V collagen humoral immunity in lung transplant primary graft dysfunction.

Primary graft dysfunction (PGD) is a major complication following lung transplantation. We reported that anti-type V collagen (col(V)) T cell immunity was strongly associated with PGD. However, the role of preformed anti-col(V) Abs and their potential target in PGD are unknown. Col(V) immune serum, purified IgG or B cells from col(V) immune rats were transferred to WKY rat lung isograft recipients followed by assessments of lung pathology, cytokines, and PaO(2)/FiO(2), an index of lung dysfunction in PGD. Immune serum, purified IgG, and B cells all induced pathology consistent with PGD within 4 days posttransfer; up-regulated IFN-gamma, TNF-alpha, and IL-1beta locally; and induced significant reductions in PaO(2)/FiO(2). Depleting anti-col(V) Abs before transfer demonstrated that IgG2c was a major subtype mediating injury. Confocal microscopy revealed strong apical col(V) expression on lung epithelial, but not endothelial cells; which was consistent with the ability of col(V) immune serum to induce complement-dependent cytotoxicity only in the epithelial cells. Examination of plasma from patients with or without PGD revealed that higher levels of preformed anti-col(V) Abs were strongly associated with PGD development. This study demonstrates a major role for anti-col(V) humoral immunity in PGD, and identifies the airway epithelium as a target in PGD.

Th-17, monokines, collagen type V, and primary graft dysfunction in lung transplantation.

RATIONALE: The pathogenesis of primary graft dysfunction (PGD), a serious complication of lung transplantation, is poorly understood. Human studies and rodent models have shown that collagen type V (col[V]), stimulates IL-17-dependent cellular immunity after lung transplantation. OBJECTIVES: To determine whether patients with end-stage lung disease develop pretransplant col(V)-specific cellular immunity, and if so, the impact of this response on PGD. METHODS: Trans-vivo delayed-type hypersensitivity (TV-DTH) assays were used to evaluate memory T-cell responses to col(V) in 55 patients awaiting lung transplantation. Pa(O(2))/Fi(O(2)) index data were used to assess PGD. Univariate risk factor analysis was performed to identify variables associated with PGD. Rats immunized with col(V) or irrelevant antigen underwent lung isografting to determine if prior anti-col(V) immunity triggers PGD in the absence of alloreactivity. MEASUREMENTS AND MAIN RESULTS: We found that 58.8% (10/17) of patients with idiopathic pulmonary fibrosis, and 15.8% (6/38) of patients without idiopathic pulmonary fibrosis tested while on the wait list for a lung transplant were col(V) DTH positive. Col(V) reactivity was CD4(+) T-cell and monocyte mediated, and dependent on IL-17, IL-1beta, and tumor necrosis factor (TNF)-alpha. Pa(O(2))/Fi(O(2)) indices were impaired significantly 6-72 hours after transplantation in col(V)-reactive versus nonreactive patients. Univariate risk factor analysis identified only preoperative TV-DTH to col(V) and ischemic time as predictors of PGD. Finally, in a rat lung isograft model, col(V) sensitization resulted in significantly lower Pa(O(2))/Fi(O(2)), increased local TNF-alpha and IL-1beta production, and a moderate-to-severe bronchiolitis/vasculitis when compared with control isografts. CONCLUSIONS: The data suggest that activation of innate immunity by col(V)-specific Th-17 memory cells represents a novel pathway to PGD after lung transplantation.

A perpetual switching system in pulmonary capillaries.

Of the 300 billion capillaries in the human lung, a small fraction meet normal oxygen requirements at rest, with the remainder forming a large reserve. The maximum oxygen demands of the acute stress response require that the reserve capillaries are rapidly recruited. To remain primed for emergencies, the normal cardiac output must be parceled throughout the capillary bed to maintain low opening pressures. The flow-distributing system requires complex switching. Because the pulmonary microcirculation contains contractile machinery, one hypothesis posits an active switching system. The opposing hypothesis is based on passive switching that requires no regulation. Both hypotheses were tested ex vivo in canine lung lobes. The lobes were perfused first with autologous blood, and capillary switching patterns were recorded by videomicroscopy. Next, the vasculature of the lobes was saline flushed, fixed by glutaraldehyde perfusion, flushed again, and then reperfused with the original, unfixed blood. Flow patterns through the same capillaries were recorded again. The 16-min-long videos were divided into 4-s increments. Each capillary segment was recorded as being perfused if at least one red blood cell crossed the entire segment. Otherwise it was recorded as unperfused. These binary measurements were made manually for each segment during every 4 s throughout the 16-min recordings of the fresh and fixed capillaries (>60,000 measurements). Unexpectedly, the switching patterns did not change after fixation. We conclude that the pulmonary capillaries can remain primed for emergencies without requiring regulation: no detectors, no feedback loops, and no effectors-a rare system in biology. NEW & NOTEWORTHY The fluctuating flow patterns of red blood cells within the pulmonary capillary networks have been assumed to be actively controlled within the pulmonary microcirculation. Here we show that the capillary flow switching patterns in the same network are the same whether the lungs are fresh or fixed. This unexpected observation can be successfully explained by a new model of pulmonary capillary flow based on chaos theory and fractal mathematics.

Lung transplant ischemia reperfusion injury: metalloprotease inhibition down-regulates exposure of type V collagen, growth-related oncogene-induced neutrophil chemotaxis, and tumor necrosis factor-alpha expression.

BACKGROUND: Immunity to type V collagen [col(V)] contributes to lung transplant rejection. Matrix metalloproteases (MMPs), which are induced by transplant-related ischemia-reperfusion injury (IRI), could expose col(V) and regulate local IRI-induced inflammation. METHODS: To test the hypothesis that MMPs induce col(V) exposure and inflammation, Wistar-Kyoto rats were treated with the MMP inhibitor, COL-3, before inducing lung IRI without transplantation, and in parallel studies, Wistar-Kyoto lung donor and recipients were treated with COL-3 pre- and postisograft lung transplantation. RESULTS: Ischemia-reperfusion injury induced growth-related oncogene/CINC-1-dependent neutrophil influx, and up-regulated tumor necrosis factor-alpha. MMP2 and MMP9, induced at 4 and 24 hr after IRI, respectively, were associated with detection of antigenic col(V) in bronchoalveolar lavage and lung interstitium because of MMP-mediated matrix degradation. MMP-inhibitor treatment significantly reduced polymorphonuclear leukocytes, growth-related oncogene/CINC-1, and tumor necrosis factor-alpha; abrogated MMP-9 expression; and resulted in lower levels of antigenic col(V) in bronchoalveolar lavage. In the lung transplant model, inhibiting MMPs in the donor before lung harvest and in the recipient after lung transplantation resulted in improved oxygenation and diminished polymorphonuclear leukocyte influx into the isograft. CONCLUSION: MMP inhibition may be a potential therapy to prevent release of antigenic col(V) and ameliorate IRI in the transplant recipient.

A comparison of single-dose caudal clonidine, morphine, or hydromorphone combined with ropivacaine in pediatric patients undergoing ureteral reimplantation.

BACKGROUND: Caudal blockade is a common technique for pediatric postoperative analgesia. While safe and effective, caudal opioids are associated with troublesome side effects. Caudal clonidine may offer significant analgesic benefits. We prospectively compared the analgesic, side effect, and rehabilitation profiles of caudal clonidine, hydromorphone, or morphine in a group of 60 pediatric patients undergoing ureteral reimplantation. METHODS: Patients aged 6 mo to 6 yr were evenly and randomly enrolled in a double-blind manner. Patients received a single caudal dose of 2 mcg/kg of clonidine, 10 mcg/kg of hydromorphone, or 50 mcg/kg of morphine, combined with 1.0 mL/kg of 0.2% ropivacaine with epinephrine. After sevoflurane in oxygen/air anesthesia, all subjects received proxy nurse-controlled analgesia with morphine. Postoperative pain intensity, use of IV morphine, and side effects were assessed during the first 24 h. Oral intake and discharge home were recorded. RESULTS: Caudal clonidine resulted in less postoperative nausea and vomiting (P = 0.01) and pruritus (P = 0.007) than did caudal hydromorphone or caudal morphine. Caudal morphine produced more sustained initial analgesia than did caudal clonidine (P = 0.02). No difference was observed in pain scores, total morphine use, time to first oral intake or discharge home. No postoperative respiratory depression, excessive sedation, hypotension, or bradycardia was identified. CONCLUSIONS: Although caudal morphine may result in more sustained initial analgesia, caudal clonidine combined with nurse-controlled analgesia appears to provide comparable analgesia with fewer side effects. Based on these results, the use of caudal clonidine may be superior to caudal opioids after pediatric ureteral reimplantation.

Cavopulmonary assist in the neonate: an alternative strategy for single-ventricle palliation.

BACKGROUND: Cavopulmonary blood flow, rather than a systemic arterial source of pulmonary blood flow, stabilizes Norwood physiology. We hypothesized that pump-assisted cavopulmonary diversion would yield stable pulmonary and systemic hemodynamics in the neonate. This was tested in a newborn animal model of total cavopulmonary diversion and univentricular Fontan circulation. METHODS: Lambs (n = 13; mean weight, 5.6 +/- 1.5 kg; mean age, 6.8 +/- 4.0 days) were anesthetized and mechanically ventilated. Baseline hemodynamic parameters were measured. Total cavopulmonary diversion was performed with bicaval venous-to-main pulmonary artery cannulation. A miniature centrifugal pump was used to assist cavopulmonary flow. Support was titrated to normal physiologic parameters. Hemodynamic data, arterial blood gases, and lactate values were measured for 8 hours. Baseline, 1-hour, and 8-hour time points were compared by using analysis of variance. RESULTS: All animals remained stable without the use of volume loading, inotropic support, or pulmonary vasodilator therapy. Cardiac index, systemic arterial pressure, left atrial pressure, and lactate values were similar to baseline values 8 hours after surgery. Mean pulmonary arterial pressure and pulmonary vascular resistance were modestly increased 8 hours after surgery. Mean arterial pH, Po(2), and Pco(2) values remained stable throughout the study. CONCLUSIONS: Cavopulmonary assist is feasible in a neonatal animal model of total cavopulmonary diversion and univentricular Fontan circulation with acceptable pulmonary arterial pressures and without altering regional volume distribution or cardiac output. Pump-assisted cavopulmonary diversion, in combination with Norwood aortic arch reconstruction, could solve several major problems associated with a systemic shunt-dependent univentricular circulation, including hypoxemia, impaired diastolic coronary perfusion, and ventricular volume overload.

Pulmonary capillaries are recruited during pulsatile flow.

Capillaries recruit when pulmonary arterial pressure rises. The duration of increased pressure imposed in such experiments is usually on the order of minutes, although recent work shows that the recruitment response can occur in <4 s. In the present study, we investigate whether the brief pressure rise during cardiac systole can also cause recruitment and whether the recruitment is maintained during diastole. To study these basic aspects of pulmonary capillary hemodynamics, isolated dog lungs were pump perfused alternately by steady flow and pulsatile flow with the mean arterial and left atrial pressures held constant. Several direct measurements of capillary recruitment were made with videomicroscopy. The total number and total length of perfused capillaries increased significantly during pulsatile flow by 94 and 105%, respectively. Of the newly recruited capillaries, 92% were perfused by red blood cells throughout the pulsatile cycle. These data provide the first direct account of how the pulmonary capillaries respond to pulsatile flow by showing that capillaries are recruited during the systolic pulse and that, once open, the capillaries remain open throughout the pulsatile cycle.

Heterogeneous capillary recruitment among adjoining alveoli.

Pulmonary capillaries recruit when microvascular pressure is raised. The details of the relationship between recruitment and pressure, however, are controversial. There are data supporting 1). gradual homogeneous recruitment, 2). sudden and complete recruitment, and 3). heterogeneous recruitment. The present study was designed to determine whether alveolar capillary networks recruit in a variety of ways or whether one model predominates. In isolated, pump-perfused canine lung lobes, fields of six neighboring alveoli were recorded with video microscopy as pulmonary venous pressure was raised from 0 to 40 mmHg in 5-mmHg increments. The largest group of alveoli (42%) recruited gradually. Another group (33%) recruited suddenly (sheet flow). Half of the neighborhoods had at least one alveolus that paradoxically derecruited when pressure was increased, even though neighboring alveoli continued to recruit capillaries. At pulmonary venous pressures of 40 mmHg, 86% of the alveolar-capillary networks were not fully recruited. We conclude that the pattern of recruitment among neighboring alveoli is complex, is not homogeneous, and may not reach full recruitment, even under extreme pressures.

Blood flow switching among pulmonary capillaries is decreased during high hematocrit.

Pulmonary capillary perfusion within a single alveolar wall continually switches among segments, even when large-vessel hemodynamics are constant. The mechanism is unknown. We hypothesize that the continually varying size of plasma gaps between individual red blood cells affects the likelihood of capillary segment closure and the probability of cells changing directions at the next capillary junction. We assumed that an increase in hematocrit would decrease the average distance between red blood cells, thereby decreasing the switching at each capillary junction. To test this idea, we observed 26 individual alveolar capillary networks by using videomicroscopy of excised canine lung lobes that were perfused first at normal hematocrit (31-43%) and then at increased hematocrit (51-62%). The number of switches decreased by 38% during increased hematocrit (P < 0.01). These results support the idea that a substantial part of flow switching among pulmonary capillaries is caused by the particulate nature of blood passing through a complex network of tubes with continuously varying hematocrit.

Selective endothelin-A receptor blockade attenuates endotoxin-induced pulmonary hypertension and pulmonary vascular dysfunction.

Endothelin-1 is a potent mediator of sepsis-induced pulmonary hypertension (PH). The pulmonary vascular effects of selective blockade of endothelin receptor subtype A (ETAR) during endotoxemia remain unknown. We hypothesized that selective ETAR antagonism attenuates endotoxin-induced PH and improves pulmonary artery (PA) vasoreactivity. Adult male Sprague-Dawley rats (250-450 g) received lipopolysaccharide (LPS; Salmonella typhimurium; 20 mg/kg intraperitoneally) or vehicle 6 hours before hemodynamic assessment and tissue harvest. The selective ETAR antagonist sitaxsentan (10 or 20 mg/kg) or vehicle was injected intravenously 3 hours after receipt of LPS. Right ventricular systolic pressure, mean arterial pressure (MAP), cardiac output (CO), oxygenation (P/F ratio), and serum bicarbonate were measured. Bronchoalveolar lavage (BAL) cell differential and lung wet-to-dry ratios were obtained. Endothelium-dependent and endothelium-independent vasorelaxations were determined in isolated PA rings. PA interleukin (IL)-1ß, IL-6, tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) were measured. LPS caused PH, decreased MAP, CO, and serum bicarbonate, and increased PA IL-1ß, IL-6, TNF-α, and iNOS mRNA. Sitaxsentan attenuated sepsis-induced PH and increased MAP. The P/F ratio, CO, serum bicarbonate, and BAL neutrophilia were not affected by sitaxsentan. In isolated PA rings, while not affecting phenylephrine-induced vasocontraction or endothelium-dependent relaxation, sitaxsentan dose-dependently attenuated LPS-induced alterations in endothelium-independent relaxation. PA cytokine mRNA levels were not significantly attenuated by ETAR blockade. We conclude that ETAR blockade attenuates endotoxin-induced alterations in systemic and PA pressures without negatively affecting oxygenation. This protective effect appears to be mediated not by attenuation of sepsis-induced cardiac dysfunction, acidosis, or alveolar inflammation but rather by improved endothelium-independent vasorelaxation.

Loss of cystic fibrosis transmembrane conductance regulator impairs lung endothelial cell barrier function and increases susceptibility to microvascular damage from cigarette smoke.

Abnormal lung microvascular endothelial vascular barrier function may contribute to pulmonary inflammation, such as that occurring during inhalation of cigarette smoke (CS). Cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in both epithelial and endothelial cells, regulates the organization of tight junctions between epithelial cells and has also been implicated in the transport of sphingosine-1 phosphate (S1P), a vascular barrier-enhancing sphingolipid. Because CS has been shown to affect CFTR function, we hypothesized that CFTR function contributes to lung endothelial cell barrier and that CFTR dysfunction worsens CS-induced injury. CFTR inhibitors GlyH-101 or CFTRinh172 caused a dose-dependent increase in pulmonary or bronchial endothelial monolayer permeability, which peaked after 4 hours. CFTR inhibition was associated with both intercellular gaps and actin stress fiber formation compared with vehicle-treated cells. Increasing endothelial S1P, either by exogenous treatment or by inhibition of its degradation, significantly improved the barrier function in CFTR-inhibited monolayers. Both cultured lung endothelia and the lung microcirculation visualized in vivo with intravital two-photon imaging of transgenic mice deficient in CFTR showed that CFTR dysfunction increased susceptibility to CS-induced permeability. These results suggested that CFTR function might be required for lung endothelial barrier, including adherence junction stability. Loss of CFTR function, especially concomitant to CS exposure, might promote lung inflammation by increasing endothelial cell permeability, which could be ameliorated by S1P.