TLR4 through IFN-ß promotes low molecular mass hyaluronan-induced neutrophil apoptosis.

Intratracheal administration of low molecular mass (LMM) hyaluronan (200 kDa) results in greater neutrophil infiltration in the lungs of TLR4(-/-) mice compared with that in wild-type mice. In general, enhanced neutrophil infiltration in tissue is due to cell influx; however, neutrophil apoptosis also plays an important role. We have assessed the effects of TLR4 in the regulation of neutrophil apoptosis in response to administration of LMM hyaluronan. We found that apoptosis of inflammatory neutrophils is impaired in TLR4(-/-) mice, an effect that depends upon the IFN-ß-mediated TRAIL/TRAILR system. IFN-ß levels were decreased in LMM hyaluronan-treated TLR4-deficient neutrophils. The treatment of inflammatory neutrophils with IFN-ß enhanced the levels of TRAIL and TRAILR 2. LMM hyaluronan-induced inflammatory neutrophil apoptosis was substantially prevented by anti-TRAIL neutralizing mAb. We conclude that decreased IFN-ß levels decrease the activity of the TRAIL/TRAILR system in TLR4-deficient neutrophils, leading to impaired apoptosis of neutrophils and resulting in abnormal accumulation of neutrophils in the lungs of LMM hyaluronan-treated mice. Thus, TLR4 plays a novel homeostatic role in noninfectious lung inflammation by accelerating the elimination of inflammatory neutrophils.

TLR4 is a negative regulator in noninfectious lung inflammation.

Low m.w. hyaluronan (LMW HA) has been shown to elicit the expression of proinflammatory cytokines and chemokines in various cells in vitro. However, the effects of this molecule in vivo are unknown. In this study, we report that intratracheal administration of LMW HA (200 kDa) causes inflammation in mouse lung. A lack of TLR4 is associated with even stronger inflammatory response in the lung as shown by increased neutrophil counts and elevated cytokine and chemokine concentrations. We also demonstrate that TLR4 anti-inflammatory signaling is dependent upon a MyD88-independent pathway. TLR4-mediated IL-1R antagonist production plays a negative regulatory role in LMW HA (200 kDa) induced lung inflammation. These data provide a molecular level explanation for the function of TLR4 in LMW HA (200 kDa)-induced lung inflammation, as inhibition of the beta form of pro-IL-1 promotes an anti-inflammatory response.

Silencing of sodium-hydrogen exchanger 1 attenuates the proliferation, hypertrophy, and migration of pulmonary artery smooth muscle cells via E2F1.

We previously found that deficiency of the sodium-hydrogen exchanger 1 (NHE1) gene prevented hypoxia-induced pulmonary hypertension and vascular remodeling in mice, which were accompanied by a significantly reduced proliferation of pulmonary artery smooth muscle cells (PASMCs), and which decreased the medial-wall thickness of pulmonary arteries. That finding indicated the involvement of NHE1 in the proliferation and hypertrophy of PASMCs, but the underlying mechanism was not fully understood. To define the mechanism by which the inhibition of NHE1 decreases hypoxic pulmonary hypertension and vascular remodeling, we investigated the role of E2F1, a nuclear transcription factor, in silencing the NHE1 gene-induced inhibition of the proliferation, hypertrophy, and migration of human PASMCs. We found that: (1) silencing of NHE1 by short, interfering RNA (siRNA) significantly inhibited PASMC proliferation and cell cycle progression, decreased hypoxia-induced hypertrophy (in terms of cell size and protein/DNA ratio) and migration (in terms of the wound-healing and migration chamber assays); (2) hypoxia induced the expression of E2F1, which was reversed by NHE1 siRNA; and (3) the overexpression of E2F1 blocked the inhibitory effect of NHE1 siRNA on the proliferation, hypertrophy, and migration of PASMCs. The present study determined that silencing the NHE1 gene significantly inhibited the hypoxia-induced proliferation, hypertrophy, and migration of human PASMCs via repression of the nuclear transcription factor E2F1. This study revealed a novel mechanism underlying the regulation of hypoxic pulmonary hypertension and vascular remodeling via NHE1.

Heparin inhibits pulmonary artery smooth muscle cell proliferation through guanine nucleotide exchange factor-H1/RhoA/Rho kinase/p27.

Ras homolog gene family member A (RhoA) through Rho kinase kinase (ROCK), one of its downstream effectors, regulates a wide range of cell physiological functions, including vascular smooth muscle cell (SMC) proliferation, by degrading cyclin-dependent kinase inhibitor, p27. Our previous studies found that heparin inhibition of pulmonary artery SMC (PASMC) proliferation and pulmonary hypertension was dependent on p27 up-regulation. To investigate whether ROCK, a regulator of p27, is involved in regulation of heparin inhibition of PASMC proliferation, we analyzed ROCK expression in the lungs from mice and from human PASMCs exposed to hypoxia, and investigated the effect of ROCK expression in vitro by RhoA cDNA transfection. We also investigated the effect of guanine nucleotide exchange factor (GEF)-H1, an upstream regulator of RhoA, on heparin inhibition of PASMC proliferation by GEF-H1 cDNA transfection. We found that: (1) hypoxia increased ROCK expression in mice and PASMCs; (2) overexpression of RhoA diminished the inhibitory effect of heparin on PASMC proliferation and down-regulated p27 expression; and (3) overexpression of GEF-H1 negated heparin inhibition of PASMC proliferation, which was accompanied by increased GTP-RhoA and decreased p27. This study demonstrates that the RhoA/ROCK pathway plays an important role in heparin inhibition on PASMC proliferation, and reveals that heparin inhibits PASMC proliferation through GEF-H1/RhoA/ROCK/p27 signaling pathway, by down-regulating GEF-H1, RhoA, and ROCK, and then up-regulating p27.

Hypoxia does neither stimulate pulmonary artery endothelial cell proliferation in mice and rats with pulmonary hypertension and vascular remodeling nor in human pulmonary artery endothelial cells.

BACKGROUND: Hypoxia results in pulmonary hypertension and vascular remodeling due to induction of pulmonary artery cell proliferation. Besides pulmonary artery smooth muscle cells, pulmonary artery endothelial cells (PAECs) are also involved in the development of pulmonary hypertension, but the effect of hypoxia on PAEC proliferation has not been completely understood. METHODS: We investigated PAEC proliferation in mice and rats with hypoxia-induced pulmonary hypertension and vascular remodeling as well as in human PAECs under hypoxia. RESULTS AND CONCLUSION: We did not find significant PAEC proliferation in chronically hypoxic rats or mice. There was a slight decrease in proliferation in mice and rats with pulmonary hypertension and vascular remodeling. We also did not find significant human PAEC proliferation and cell cycle progression under different levels of oxygen (1, 2, 3, 5 and 10%) for one day, although the same conditions of hypoxia induced significant proliferation and cell cycle progression in pulmonary artery smooth muscle cells and pulmonary artery fibroblasts. Exposure to hypoxia for 7 days also did not increase PAEC proliferation. These results demonstrated that hypoxia alone is not a stimulus to PAEC proliferation in vivo and in vitro. The present study provides a novel role for PAECs in hypoxia-induced pulmonary hypertension and vascular remodeling.

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression.

OBJECTIVE: The mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling. METHODS: Vascular remodeling was induced in CD44(+/+) and CD44(-/-) mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs. RESULTS: Transluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44(-/-) mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor. CONCLUSIONS: LMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression.

Effect of chemokine receptor CXCR4 on hypoxia-induced pulmonary hypertension and vascular remodeling in rats.

BACKGROUND: CXCR4 is the receptor for chemokine CXCL12 and reportedly plays an important role in systemic vascular repair and remodeling, but the role of CXCR4 in development of pulmonary hypertension and vascular remodeling has not been fully understood. METHODS: In this study we investigated the role of CXCR4 in the development of pulmonary hypertension and vascular remodeling by using a CXCR4 inhibitor AMD3100 and by electroporation of CXCR4 shRNA into bone marrow cells and then transplantation of the bone marrow cells into rats. RESULTS: We found that the CXCR4 inhibitor significantly decreased chronic hypoxia-induced pulmonary hypertension and vascular remodeling in rats and, most importantly, we found that the rats that were transplanted with the bone marrow cells electroporated with CXCR4 shRNA had significantly lower mean pulmonary pressure (mPAP), ratio of right ventricular weight to left ventricular plus septal weight (RV/(LV+S)) and wall thickness of pulmonary artery induced by chronic hypoxia as compared with control rats. CONCLUSIONS: The hypothesis that CXCR4 is critical in hypoxic pulmonary hypertension in rats has been demonstrated. The present study not only has shown an inhibitory effect caused by systemic inhibition of CXCR4 activity on pulmonary hypertension, but more importantly also has revealed that specific inhibition of the CXCR4 in bone marrow cells can reduce pulmonary hypertension and vascular remodeling via decreasing bone marrow derived cell recruitment to the lung in hypoxia. This study suggests a novel therapeutic approach for pulmonary hypertension by inhibiting bone marrow derived cell recruitment.

Long-term exposure to hypoxia inhibits tumor progression of lung cancer in rats and mice.

BACKGROUND: Hypoxia has been identified as a major negative factor for tumor progression in clinical observations and in animal studies. However, the precise role of hypoxia in tumor progression has not been fully explained. In this study, we extensively investigated the effect of long-term exposure to hypoxia on tumor progression in vivo. METHODS: Rats bearing transplanted tumors consisting of A549 human lung cancer cells (lung cancer tumor) were exposed to hypoxia for different durations and different levels of oxygen. The tumor growth and metastasis were evaluated. We also treated A549 lung cancer cells (A549 cells) with chronic hypoxia and then implanted the hypoxia-pretreated cancer cells into mice. The effect of exposure to hypoxia on metastasis of Lewis lung carcinoma in mice was also investigated. RESULTS: We found that long-term exposure to hypoxia a) significantly inhibited lung cancer tumor growth in xenograft and orthotopic models in rats, b) significantly reduced lymphatic metastasis of the lung cancer in rats and decreased lung metastasis of Lewis lung carcinoma in mice, c) reduced lung cancer cell proliferation and cell cycle progression in vitro, d) decreased growth of the tumors from hypoxia-pretreated A549 cells, e) decreased Na+-K+ ATPase α1 expression in hypoxic lung cancer tumors, and f) increased expression of hypoxia inducible factors (HIF1α and HIF2α) but decreased microvessel density in the lung cancer tumors. In contrast to lung cancer, the growth of tumor from HCT116 human colon cancer cells (colon cancer tumor) was a) significantly enhanced in the same hypoxia conditions, accompanied by b) no significant change in expression of Na+-K+ ATPase α1, c) increased HIF1α expression (no HIF2α was detected) and d) increased microvessel density in the tumor tissues. CONCLUSIONS: This study demonstrated that long-term exposure to hypoxia repressed tumor progression of the lung cancer from A549 cells and that decreased expression of Na+-K+ ATPase was involved in hypoxic inhibition of tumor progression. The results from this study provide new insights into the role of hypoxia in tumor progression and therapeutic strategies for cancer treatment.

Anti-proliferative effects of O-acyl-low-molecular-weight heparin derivatives on bovine pulmonary artery smooth muscle cells.

Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri-n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 (O-hexanoylated LMWHP) and at a carbon chain length 12-18 (O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity.

Interleukin-6 overexpression induces pulmonary hypertension.

Inflammatory cytokine interleukin (IL)-6 is elevated in the serum and lungs of patients with pulmonary artery hypertension (PAH). Several animal models of PAH cite the potential role of inflammatory mediators. We investigated role of IL-6 in the pathogenesis of pulmonary vascular disease. Indices of pulmonary vascular remodeling were measured in lung-specific IL-6-overexpressing transgenic mice (Tg(+)) and compared to wild-type (Tg(-)) controls in both normoxic and chronic hypoxic conditions. The Tg(+) mice exhibited elevated right ventricular systolic pressures and right ventricular hypertrophy with corresponding pulmonary vasculopathic changes, all of which were exacerbated by chronic hypoxia. IL-6 overexpression increased muscularization of the proximal arterial tree, and hypoxia enhanced this effect. It also reproduced the muscularization and proliferative arteriopathy seen in the distal arteriolar vessels of PAH patients. The latter was characterized by the formation of occlusive neointimal angioproliferative lesions that worsened with hypoxia and were composed of endothelial cells and T-lymphocytes. IL-6-induced arteriopathic changes were accompanied by activation of proangiogenic factor, vascular endothelial growth factor, the proproliferative kinase extracellular signal-regulated kinase, proproliferative transcription factors c-MYC and MAX, and the antiapoptotic proteins survivin and Bcl-2 and downregulation of the growth inhibitor transforming growth factor-beta and proapoptotic kinases JNK and p38. These findings suggest that IL-6 promotes the development and progression of pulmonary vascular remodeling and PAH through proproliferative antiapoptotic mechanisms.

Gadolinium-enhanced magnetic resonance angiography for pulmonary embolism: a multicenter prospective study (PIOPED III).

BACKGROUND: The accuracy of gadolinium-enhanced magnetic resonance pulmonary angiography and magnetic resonance venography for diagnosing pulmonary embolism has not been determined conclusively. OBJECTIVE: To investigate performance characteristics of magnetic resonance angiography, with or without magnetic resonance venography, for diagnosing pulmonary embolism. DESIGN: Prospective, multicenter study from 10 April 2006 to 30 September 2008. SETTING: 7 hospitals and their emergency services. PATIENTS: 371 adults with diagnosed or excluded pulmonary embolism. MEASUREMENTS: Sensitivity, specificity, and likelihood ratios were measured by comparing independently read magnetic resonance imaging with the reference standard for diagnosing pulmonary embolism. Reference standard diagnosis or exclusion was made by using various tests, including computed tomographic angiography and venography, ventilation-perfusion lung scan, venous ultrasonography, d-dimer assay, and clinical assessment. RESULTS: Magnetic resonance angiography, averaged across centers, was technically inadequate in 25% of patients (92 of 371). The proportion of technically inadequate images ranged from 11% to 52% at various centers. Including patients with technically inadequate images, magnetic resonance angiography identified 57% (59 of 104) with pulmonary embolism. Technically adequate magnetic resonance angiography had a sensitivity of 78% and a specificity of 99%. Technically adequate magnetic resonance angiography and venography had a sensitivity of 92% and a specificity of 96%, but 52% of patients (194 of 370) had technically inadequate results. LIMITATION: A high proportion of patients with suspected embolism was not eligible or declined to participate. CONCLUSION: Magnetic resonance pulmonary angiography should be considered only at centers that routinely perform it well and only for patients for whom standard tests are contraindicated. Magnetic resonance pulmonary angiography and magnetic resonance venography combined have a higher sensitivity than magnetic resonance pulmonary angiography alone in patients with technically adequate images, but it is more difficult to obtain technically adequate images with the 2 procedures.

JNK activation is responsible for mucus overproduction in smoke inhalation injury.

BACKGROUND: Increased mucus secretion is one of the important characteristics of the response to smoke inhalation injuries. We hypothesized that gel-forming mucins may contribute to the increased mucus production in a smoke inhalation injury. We investigated the role of c-Jun N-terminal kinase (JNK) in modulating smoke-induced mucus secretion. METHODS: We intubated mice and exposed them to smoke from burning cotton for 15 min. Their lungs were then isolated 4 and 24 h after inhalation injury. Three groups of mice were subjected to the smoke inhalation injury: (1) wild-type (WT) mice, (2) mice lacking JNK1 (JNK1-/- mice), and (3) WT mice administered a JNK inhibitor. The JNK inhibitor (SP-600125) was injected into the mice 1 h after injury. RESULTS: Smoke exposure caused an increase in the production of mucus in the airway epithelium of the mice along with an increase in MUC5AC gene and protein expression, while the expression of MUC5B was not increased compared with control. We found increased MUC5AC protein expression in the airway epithelium of the WT mice groups both 4 and 24 h after smoke inhalation injury. However, overproduction of mucus and increased MUC5AC protein expression induced by smoke inhalation was suppressed in the JNK inhibitor-treated mice and the JNK1 knockout mice. Smoke exposure did not alter the expression of MUC1 and MUC4 proteins in all 3 groups compared with control. CONCLUSION: An increase in epithelial MUC5AC protein expression is associated with the overproduction of mucus in smoke inhalation injury, and that its expression is related on JNK1 signaling.

High MW hyaluronan inhibits smoke inhalation-induced lung injury and improves survival.

BACKGROUND AND OBJECTIVE: High MW hyaluronan (HMW HA) as opposed to low MW hyaluronan (LMW HA) has been shown to have anti-inflammatory and anti-apoptotic effects. We hypothesized that treatment with HMW HA would block smoke inhalation lung injury by inhibiting smoke-induced lung inflammation and airway epithelial cell apoptosis. METHODS: Anesthetized, intubated male rats were randomly allocated to either control or smoke inhalation injury groups. Rats were treated with 3-mL subcutaneous normal saline solution (sham) or LMW HA (35 kDa) or HMW HA (1600 kDa) 18 h before exposure to 15 min of cotton smoke (n = 5 each). Rats were also treated post smoke inhalation with 1600 kDa HA by intra-peritoneal injection (3 mL) or intra-tracheal nebulization (200 µL). Lung neutrophil infiltration, airway apoptosis, airway mucous plugging and lung injury were assessed 4 h after smoke inhalation injury. RESULTS: Rats pretreated with 1600 kDa HA had significantly less smoke-induced neutrophil infiltration, lung oedema, airway apoptosis and mucous plugging. Pretreatment with 35 kDa HA, in contrast, increased smoke-induced neutrophil infiltration and lung injury score. Intra-tracheal administration of a single dose 1600 kDa HA, but not intra-peritoneal injection, significantly improved survival post smoke inhalation. CONCLUSIONS: High MW hyaluronan (1600 kDa) may prove to be a beneficial therapy for smoke inhalation through inhibition of smoke-induced inflammation, lung oedema, airway epithelial cell apoptosis and airway mucous plugging.

Growth inhibition of bovine pulmonary artery smooth muscle cells following long-term heparin treatment.

Heparin (HP) inhibits pulmonary artery smooth muscle cell (PASMC) growth in vitro and vascular remodeling in vivo. Bârzu et al. (1994) suggested that the antiproliferative effect of HP on rat aortic smooth muscle cell in vitro diminishes with prolonged exposure to heparin. We exposed cultured bovine PASMC (BPASMC) to prolonged pretreatment with 20 microg/ml of 0-hexanoylated HP from passages 3 to13 and compared them to control (no pretreatment) cultures of identical passages. The pretreated BPASMC and control groups were growth arrested for 48 h, followed by treatment of 0-hexanoylated HP at different doses. On day 5, the growth inhibition of BPASMC was determined. The percent inhibition by 1 microg/ml of 0-hexanoylated HP was 46 +/- 14% versus 62 +/- 13%, for control and pretreated BPASMC, respectively. At 10 microg/ml the inhibition was 62 +/- 7% versus 84 +/- 6%. For 100 microg/ml the inhibition increased to 92 +/- 5% versus 100% and at 200 microg/ml the inhibition was 95 +/- 3% versus 100%. BPASMC (with or without preexposure to 0-hexanoylated HP), at passage 13, were sensitive to the growth inhibitory effect of 0-hexanoylated HP with no significant difference among the groups (95 +/- 3% inhibition vs. 100% for pretreated BPASMC). We found that 0-hexanoylated HP-induced necrosis as shown by flow cytometry and only minor apoptosis. Caspase-3 and PARP detection was insignificant between the groups. In summary, no cell subpopulation at long-term treatment exhibited resistance to 0-hexanoylated HP. The HP antiproliferative effect on SMC is potentially important in defining new approaches to the treatment of the remodeled vasculature of pulmonary hypertension. Liss, Inc.

Multidetector computed tomography for acute pulmonary embolism.

BACKGROUND: The accuracy of multidetector computed tomographic angiography (CTA) for the diagnosis of acute pulmonary embolism has not been determined conclusively. METHODS: The Prospective Investigation of Pulmonary Embolism Diagnosis II trial was a prospective, multicenter investigation of the accuracy of multidetector CTA alone and combined with venous-phase imaging (CTA-CTV) for the diagnosis of acute pulmonary embolism. We used a composite reference test to confirm or rule out the diagnosis of pulmonary embolism. RESULTS: Among 824 patients with a reference diagnosis and a completed CT study, CTA was inconclusive in 51 because of poor image quality. Excluding such inconclusive studies, the sensitivity of CTA was 83 percent and the specificity was 96 percent. Positive predictive values were 96 percent with a concordantly high or low probability on clinical assessment, 92 percent with an intermediate probability on clinical assessment, and nondiagnostic if clinical probability was discordant. CTA-CTV was inconclusive in 87 of 824 patients because the image quality of either CTA or CTV was poor. The sensitivity of CTA-CTV for pulmonary embolism was 90 percent, and specificity was 95 percent. CTA-CTV was also nondiagnostic with a discordant clinical probability. CONCLUSIONS: In patients with suspected pulmonary embolism, multidetector CTA-CTV has a higher diagnostic sensitivity than does CTA alone, with similar specificity. The predictive value of either CTA or CTA-CTV is high with a concordant clinical assessment, but additional testing is necessary when the clinical probability is inconsistent with the imaging results.

The role of phosphodiesterase 3 in endotoxin-induced acute kidney injury.

BACKGROUND: Acute kidney injury frequently accompanies sepsis. Endotoxin is known to reduce tissue levels of cAMP and low levels of cAMP have been associated with renal injury. We, therefore, hypothesized that endotoxin induced renal injury by activating phosphodiesterase 3 (PDE3) which metabolizes cAMP and that amrinone an inhibitor of PDE3 would prevent the renal injury. METHODS: Animals were divided into three groups (n = 7/group): 1) Control (0.9% NaCl infusion without LPS); 2) LPS (0.9% NaCl infusion with LPS); 3) Amrinone+LPS (Amrinone infusion with LPS). Either lipopolysaccharide (LPS) or vehicle was injected via the jugular vein and the rats followed for 3 hours. We explored the expression of PDE3 isoenzymes and the concentrations of cAMP in the tissue. RESULTS: The PDE3B gene but not PDE3A was upregulated in the kidney of LPS group. Immunohistochemistry also showed that PDE3B was expressed in the distal tubule in the controls and LPS caused PDE3B expression in the proximal as well. However, PDE3A was not expressed in the kidney either in the control or LPS treated groups. Tissue level of cAMP was decreased after LPS and was associated with an increase in blood urea nitrogen, creatinine, ultrastructural proximal tubular changes, and expression of inducible nitric oxide synthase (iNOS) in the endotoxemic kidney. In septic animals the phosphodiesterase 3 inhibitor, amrinone, preserved the tissue cAMP level, renal structural changes, and attenuated the increased blood urea nitrogen, creatinine, and iNOS expression in the kidney. CONCLUSION: These findings suggest a significant role for PDE3B as an important mediator of LPS-induced acute kidney injury.

Reconstructed 4-chamber views compared with axial imaging for assessment of right ventricular enlargement on CT pulmonary angiograms.

PURPOSE: To test the hypothesis that right enlargement assessed from right ventricular/left ventricular (RV/LV) dimension ratios of computed tomographic (CT) angiograms are equivalent irrespective of whether measured on axial views or reconstructed 4-chamber views. METHODS: RV/LV dimension ratios were calculated from measurements on axial views, manually reconstructed 4-chamber views and computer generated reconstructed 4-chamber views of CT angiograms in 152 patients with PE. RESULTS: Paired readings of the axial view and manually reconstructed 4-chamber view showed agreement with RV/LV > or =1 or RV/LV <1 in 114 of 127 (89.8%). Paired readings also showed agreement in 119 of 127 (93.7%) with axial views and computer generated reconstructed 4-chamber views. The McNemar test showed no statistically significant difference between assessments of RV enlargement (RV/LV > or = 1) with any method. CONCLUSION: Right ventricular enlargement can be determined from axial views on CT angiograms, which are readily and immediately available, without obtaining 4-chamber reconstructed views.

Inhibition of HA synthase 3 mRNA expression, with a phosphodiesterase 3 inhibitor, blocks lung injury in a septic ventilated rat model.

Low-molecular-weight hyaluronan produced by hyaluronan synthase 3 (HAS3) has been shown to play a role in acute lung injury secondary to high-tidal-volume ventilation. Phosphodiesterase 3 inhibitors have been shown to decrease HAS3 expression. We hypothesized that low-molecular-weight hyaluronan (LMW HA) produced by HAS3 mediates LPS-induced lung injury in the mechanically ventilated rat and that milrinone (MIL), by blocking HAS3 mRNA expression, would prevent the injury. Rats were randomized to four groups: controls with mechanical ventilation at 7 cc/kg MV, MV+LPS, MV+MIL, and MV+LPS+MIL. Rats were intubated and ventilated without PEEP for 4 h. Lipopolysaccharide (LPS) (1 mg/kg) was infused into the arterial line 1 h prior to MV. MIL 10 microg/kg/min (or an equivalent volume of saline) was infused through the venous line at the beginning of MV. Bronchoalveolar lavage fluid (BAL) was collected after 4 h of ventilation and lungs were saved for histopathology. LPS significantly increased neutrophil infiltration and protein concentration in the BAL and augmented lung injury score on histology. MIL significantly lowered alveolar protein and neutrophil infiltration as well as lung injury in response to LPS. Furthermore, MIL decreased the mRNA expression for HAS3 and MIP2 in lung tissue and decreased the protein content in BAL. MIL, a commonly used inotropic agent, inhibited LPS-induced lung inflammation and lung injury in mechanically ventilated rats. The anti-inflammatory properties of MIL may be mediated by inhibition of HAS3 and/or MIP2 and could be beneficial in the treatment of sepsis.

Deficiency of the NHE1 gene prevents hypoxia-induced pulmonary hypertension and vascular remodeling.

RATIONALE: Our previous studies found that Na(+)/H(+) exchanger (NHE) activity played an essential role in pulmonary artery smooth muscle cell (PASMC) proliferation and in the development of hypoxia-induced pulmonary hypertension and vascular remodeling. Other investigators recently observed increased expression of the NHE isoform 1 (NHE1) gene in rodents with pulmonary hypertension induced by hypoxia. However, a causal role for the NHE1 gene in pulmonary hypertension has not been determined. OBJECTIVES: To determine the causal role of the NHE1 gene in pulmonary hypertension and vascular remodeling. METHODS: We used NHE1-null mice to define the role of the NHE1 gene in the development of pulmonary hypertension and remodeling induced by hypoxia and to delineate the NHE1 regulatory pathway. MEASUREMENTS AND MAIN RESULTS: After 2 weeks of exposure to hypoxia, in contrast to wild-type hypoxic littermates, there was no significant increase in right ventricular systolic pressure, in the ratio of right ventricular to left ventricular plus septal weight [RV/(LV + S)], or in medial wall thickness of the pulmonary arterioles in homozygous mice (NHE1(-/-)). There was a significant decrease in Rho kinase (ROCK1 and ROCK2) expression, accompanied by an increase in p27 expression in NHE1(-/-) mice. CONCLUSIONS: Our study demonstrated that deficiency of the NHE1 gene prevented the development of hypoxia-induced pulmonary hypertension and vascular remodeling in mice and revealed a novel regulatory pathway associated with NHE1 signaling.

Cyclic stretch affects pulmonary endothelial cell control of pulmonary smooth muscle cell growth.

Endothelial cells are subjected to mechanical forces in the form of cyclic stretch resulting from blood pulsatility. Pulmonary artery endothelial cells (PAECs) produce factors that stimulate and inhibit pulmonary artery smooth muscle cell (PASMC) growth. We hypothesized that PAECs exposed to cyclic stretch secrete proteins that inhibit PASMC growth. Media from PAECs exposed to cyclic stretch significantly inhibited PASMC growth in a time-dependent manner. Lyophilized material isolated from stretched PAEC-conditioned media significantly inhibited PASMC growth in a dose-dependent manner. This inhibition was reversed by trypsin inactivation, which is consistent with the relevant factor being a protein(s). To identify proteins that inhibited cell growth in conditioned media from stretched PAECs, we used proteomic techniques and found that thrombospondin (TSP)-1, a natural antiangiogenic factor, was up-regulated by stretch. In vitro, exogenous TSP-1 inhibited PASMC growth. TSP-1-blocking antibodies reversed conditioned media-induced inhibition of PASMC growth. Cyclic stretched PAECs secrete protein(s) that inhibit PASMC proliferation. TSP-1 may be, at least in part, responsible for this inhibition. The complete identification and understanding of the secreted proteome of stretched PAECs may lead to new insights into the pathophysiology of pulmonary vascular remodeling.