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1.  Increase in the growth inhibition of bovine pulmonary artery smooth muscle cells by an O-hexanoyl low-molecular-weight heparin derivative 
Carbohydrate research  2006;341(15):2607-2612.
Proliferation of pulmonary artery smooth muscle cells (PASMCs) appears to play a significant role in chronic pulmonary hypertension. The proliferation of PASMCs is strongly inhibited by some commercial heparin preparations. Heparin fragments were prepared by periodate treatment, followed by sodium borohydride reduction, to enhance potency. The tributylammonium salt of this fragmented heparin was O-acylated with hexanoic anhydride. Gradient polyacrylamide gel electrophoresis showed that the major heparin fragment contained eight disaccharide units. NMR analysis showed that approximately one hexanoyl group per disaccharide residue was present. The O-hexanoyl heparin fragments were assayed for growth inhibitory effect on bovine PASMCs in culture. This derivative was found to be more effective in growth inhibition of bovine PASMCs in culture than the heparin from which it was derived. In the future, it is envisioned that this or similar derivatives may be an effective treatment for pulmonary hypertension.
PMCID: PMC4129652  PMID: 16920092
O-Hexanoyl heparin; Pulmonary artery smooth muscle cell proliferation
2.  Comparison of 1.5 and 3.0 T for Contrast-Enhanced Pulmonary Magnetic Resonance Angiography 
In a recent multi-center trial of gadolinium contrast-enhanced magnetic resonance angiography (Gd-MRA) for diagnosis of acute pulmonary embolism (PE), two centers utilized a common MRI platform though at different field strengths (1.5T and 3T) and realized a signal-to-noise gain with the 3T platform. This retrospective analysis investigates this gain in signal-to-noise of pulmonary vascular targets.
Thirty consecutive pulmonary MRA examinations acquired on a 1.5T system at one institution were compared to 30 consecutive pulmonary MRA examinations acquired on a 3T system at a different institution. Both systems were from the same MRI manufacturer and both used the same Gd-MRA pulse sequence, although there were some protocol adjustments made due to field strength differences. Region-of-interests were manually defined on the main pulmonary artery, 4 pulmonary veins, thoracic aorta, and background lung for objective measurement of signal-to-noise, contrast-to-noise, and bolus timing bias between centers.
The 3T pulmonary MRA protocol achieved higher spatial resolution yet maintained significantly higher signal-to-noise ratio (≥ 13%, p = 0.03) in the main pulmonary vessels relative to 1.5T. There was no evidence of operator bias in bolus timing or patient hemodynamic differences between groups.
Relative to 1.5T, higher spatial resolution Gd-MRA can be achieved at 3T with a sustained or greater signal-to-noise ratio of enhanced vasculature.
PMCID: PMC3698487  PMID: 21993980
pulmonary embolism; MRI; field strength; image quality
3.  Signal quality of single dose gadobenate dimeglumine pulmonary MRA examinations exceeds quality of MRA performed with double dose gadopentetate dimeglumine 
During a recent multi-center trial assessing gadolinium (Gd)-enhanced magnetic resonance angiography (MRA) for diagnosis of acute pulmonary embolism (PE), the Food and Drug Administration announced a risk of nephrogenic sclerosing fibrosis in patients with renal insufficiency who had received intravenous Gd-based MR contrast agents. Although no patients in this trial had renal insufficiency, in cautious response to this announcement, the trial protocol was changed from an intravenous administration of 0.2 mmol/Kg of a conventional Gd-based MR contrast agent to 0.1 mmol/Kg of gadobenate dimeglumine. The study described herein compares the signal quality of pulmonary MRA performed with double dose conventional agent to single dose gadob-enate dimeglumine. This study is a retrospective analysis of data from a prospective, multicenter study in men and women ≥18 years with documented presence or absence of PE. The study was approved by the Institutional Review Board at all participating centers, and all patients provided written indication of informed consent. We performed both objective and subjective analysis of pulmonary artery image quality. Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) in the main pulmonary artery were assessed in single and double dose protocols and compared. SNR and CNR of the main PA were correlated with subjective quality assessment of main/lobar, segmental and subsegmental pulmonary arteries. Although there were individual outliers, both SNR (P = 0.01) and CNR (P = 0.008) were higher in all quartiles for examinations using gadobenate dimeglumine than with gadopentetate dimeglumine. Subjective quality of vascular signal intensity at each vessel order was significantly better for gadobenate dimeglumine (P < 0.0001), and correlated well with SNR and CNR at each order (<0.001). Because of agent high relaxivity, a single dose of gadobenate dimeglumine provides better pulmonary MRA signal quality than double dose of a conventional Gd-based MR contrast agent.
PMCID: PMC3141086  PMID: 21337023
Pulmonary MR angiography; Contrast agents; Image quality; Pulmonary embolism
4.  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.
PMCID: PMC3262694  PMID: 21454803
sodium–hydrogen exchanger 1; E2F1; PASMC proliferation; hypertrophy; hypoxia
5.  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 
Journal of Vascular Research  2011;48(6):465-475.
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.
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.
PMCID: PMC3128130  PMID: 21691120
Pulmonary artery endothelial cell; Proliferation; Hypoxia; Mice; Rats
6.  Anti-proliferative effects of O-acyl-low-molecular-weight heparin derivatives on bovine pulmonary artery smooth muscle cells 
Glycoconjugate journal  2011;28(6):419-426.
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.
PMCID: PMC3234589  PMID: 21773727
heparin; low molecular weight heparin; O-acylated; smooth muscle cells; surface plasmon resonance
7.  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.
PMCID: PMC3095924  PMID: 20558775
Rho kinase; guanine nucleotide exchange factor–H1; pulmonary artery smooth muscle cell; proliferation; heparin
8.  Factors in the technical quality of gadolinium enhanced magnetic resonance angiography for pulmonary embolism in PIOPED III 
In a multi-center trial, gadolinium enhanced magnetic resonance angiography (MRA) for diagnosis of acute pulmonary embolism (PE) had a high rate of technically inadequate images. Accordingly, we evaluated the reasons for poor quality MRA of the pulmonary arteries in these patients. We performed a retrospective analysis of the data collected in the PIOPED III study. We assessed the relationship to the proportion of examinations deemed “uninterpretable” by central readers to the clinical centers, MR equipment platform and vendors, degree of vascular opacification in different orders of pulmonary arteries; type, frequency and severity of image artifacts; patient co-morbidities, symptoms and signs; and reader characteristics. Centers, MR equipment vendor and platform, degree of vascular opacification, and motion artifacts influenced the likelihood of central reader determinations that images were “uninterpretable”. Neither the reader nor patient characteristics (age, body mass index, respiratory rate, heart rate) correlated with the likelihood of determining examinations “uninterpretable”. Vascular opacification and motion artifact are the principal factors influencing MRA interpretability. Some centers obtain better images more consistently, but the reasons for differences between centers are unclear.
PMCID: PMC3196681  PMID: 21347594
Pulmonary MR angiography; Image quality; Pulmonary embolism; Artifacts
9.  Long-term exposure to hypoxia inhibits tumor progression of lung cancer in rats and mice 
BMC Cancer  2011;11:331.
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.
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.
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.
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.
PMCID: PMC3199866  PMID: 21812995
hypoxia; tumor growth; metastasis; A549 lung cancer cells; Lewis lung carcinoma; HCT116 colon cancer cells; animals
10.  Gadolinium-Enhanced Magnetic Resonance Angiography for Pulmonary Embolism 
Annals of internal medicine  2010;152(7):434-W143.
The accuracy of gadolinium-enhanced magnetic resonance pulmonary angiography and magnetic resonance venography for diagnosing pulmonary embolism has not been determined conclusively.
To investigate performance characteristics of magnetic resonance angiography, with or without magnetic resonance venography, for diagnosing pulmonary embolism.
Prospective, multicenter study from 10 April 2006 to 30 September 2008. ( registration number: NCT00241826)
7 hospitals and their emergency services.
371 adults with diagnosed or excluded pulmonary embolism.
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 ultra-sonography, D-dimer assay, and clinical assessment.
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.
A high proportion of patients with suspected embolism was not eligible or declined to participate.
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.
Primary Funding Source
National Heart, Lung, and Blood Institute.
PMCID: PMC3138428  PMID: 20368649
11.  Effect of carboxyl-reduced heparin on the growth inhibition of bovine pulmonary artery smooth muscle cells 
Carbohydrate research  2010;345(9):1084-1087.
Heparin (HP) inhibits the proliferation of bovine pulmonary artery smooth muscle cells (BPASMC's), among other cell types in vitro. In order to develop a potential therapeutic agent to reverse vascular remodeling, we are involved in deciphering the relationship between the native HP structure and its antiproliferative potency. We have previously reported the influence of the molecular size and the effects of various O-sulfo and N-acetyl groups of HP on growth-inhibitory activity. In this study, to understand the influence of carboxyl groups in the HP structure required for endogenous activity, a chemically modified derivative of native HP was prepared by converting the carboxyl groups of hexuronic acid residues in HP to primary hydroxyl groups. This modification procedure involves the treatment of HP with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide followed by reduction with NaBH4 to yield carboxyl-reduced heparin (CR-HP). When compared to the antiproliferative potency of native HP on cultured BPASMC's at three dose levels (1, 10, and 100 μg/mL), the CR-HP showed significantly less potency at all the doses. These results suggest that hexuronic acid residues in both major and variable sequences in HP are essential for the antiproliferative properties of native HP.
PMCID: PMC2891731  PMID: 20399420
12.  Effect of chemokine receptor CXCR4 on hypoxia-induced pulmonary hypertension and vascular remodeling in rats 
Respiratory Research  2011;12(1):21.
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.
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.
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.
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.
PMCID: PMC3042398  PMID: 21294880
13.  JNK activation is responsible for mucus overproduction in smoke inhalation injury 
Respiratory Research  2010;11(1):172.
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.
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.
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.
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.
PMCID: PMC3014901  PMID: 21134294
14.  Growth Inhibition of bovine pulmonary artery smooth muscle cells Following Long Term heparin Treatment 
Journal of cellular physiology  2009;221(3):603-608.
Heparin (HP) inhibits pulmonary artery smooth-muscle cell (PASMC) growth in vitro and vascular remodeling in vivo. Barzu 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 20μg/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 hrs, followed by treatment of 0- hexanoylated HP at different doses. On day five, the growth inhibition of BPASMC was determined. The percent inhibition by 1μg/ml of 0- hexanoylated HP was 46±14% vs. 62±13%, for control and pretreated BPASMC respectively. At 10μg/ml the inhibition was 62±7% vs. 84±6%. For 100μg/ml the inhibition increased to 92±5% vs. 100% and at 200μg/ml the inhibition was 95±3% vs. 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. Caspase3 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.
PMCID: PMC2755647  PMID: 19653229
15.  Thrombospondin-1 null mice are resistant to hypoxia-induced pulmonary hypertension 
Background and objective
Chronic hypoxia induces pulmonary hypertension in mice. Smooth muscle cell hyperplasia and medial thickening characterize the vasculature of these animals. Thrombospondin-1 null (TSP-1-/-) mice spontaneously develop pulmonary smooth muscle cell hyperplasia and medial thickening. In addition, TSP-1 produced by the pulmonary endothelium inhibits pulmonary artery smooth muscle cell growth. Based on these observations we sought to describe the pulmonary vascular changes in TSP-1-/- mice exposed to chronic hypoxia.
We exposed TSP-1-/- and wild type (WT) mice to a fraction of inspired oxygen (FiO2) of 0.1 for up to six weeks. Pulmonary vascular remodeling was evaluated using tissue morphometrics. Additionally, right ventricle systolic pressures (RVSP) and right ventricular hypertrophy by right ventricle/left ventricle + septum ratios (RV/LV+S) were measured to evaluate pulmonary hypertensive changes. Finally, acute pulmonary vasoconstriction response in both TSP-1-/- and WT mice was evaluated by acute hypoxia and U-46619 (a prostaglandin F2 analog) response.
In hypoxia, TSP-1-/- mice had significantly lower RVSP, RV/LV+S ratios and less pulmonary vascular remodeling when compared to WT mice. TSP-1-/- mice also had significantly lower RVSP in response to acute pulmonary vasoconstriction challenges than their WT counterparts.
TSP-1-/- mice had diminished pulmonary vasoconstriction response and were less responsive to hypoxia-induced pulmonary hypertension than their wild type counterparts. This observation suggests that TSP-1 could play an active role in the pathogenesis of pulmonary hypertension associated with hypoxia.
PMCID: PMC2877040  PMID: 20441584
16.  Methods of Prospective Investigation of Pulmonary Embolism Diagnosis III (PIOPED III) 
Seminars in nuclear medicine  2008;38(6):462-470.
The methods of the Prospective Investigation of Pulmonary Embolism Diagnosis III (PIOPED III) are described in detail. PIOPED III is a multicenter collaborative investigation sponsored by the National Heart, Lung and Blood Institute. The purpose is to determine the accuracy of gadolinium-enhanced magnetic resonance angiography (Gd-MRA) in combination with venous phase magnetic resonance venography (Gd-MRV) for the diagnosis for acute pulmonary embolism (PE). A composite reference standard based on usual diagnostic methods for pulmonary embolism is used. All images will be read by two blinded and study-certified central readers. Patients with no PE according to the composite reference test will be randomized to undergo Gd-MRA/MRV. This will reduce the proportion of patients with negative tests at no loss in evaluation of sensitivity and specificity.
PMCID: PMC2605689  PMID: 19331840
17.  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.
PMCID: PMC2438445  PMID: 18314539
pulmonary endothelium; smooth muscle cells growth; cyclic stretch; proteomics; thrombospondin-1
18.  The role of phosphodiesterase 3 in endotoxin-induced acute kidney injury 
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.
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.
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.
These findings suggest a significant role for PDE3B as an important mediator of LPS-induced acute kidney injury.
PMCID: PMC2694814  PMID: 19486524
19.  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.
PMCID: PMC2408441  PMID: 18310478
Na+/H+ exchanger isoform 1; pulmonary hypertension; vascular remodeling; hypoxia; mouse
20.  Enlarged Right Ventricle Without Shock in Acute Pulmonary Embolism: Prognosis 
The American journal of medicine  2008;121(1):34-42.
An unsettled issue is use of thrombolytic agents in patients with acute pulmonary embolism who are hemodynamically stable, but have right ventricular enlargement.
To assess in-hospital mortality of hemodynamically stable patients with pulmonary embolism and right ventricular enlargement.
Patients were enrolled in the Prospective Investigation of Pulmonary Embolism Diagnosis II (PIOPED II). Exclusions included shock, critically ill, ventilatory support, myocardial infarction within 1 month, or ventricular tachycardia or ventricular fibrillation within 24 hours. We evaluated the ratio of the right ventricular minor axis to the left ventricular minor axis measured on transverse images during computed tomographic angiography.
Among 76 patients with right ventricular enlargement treated with anticoagulants and/or inferior vena cava filters, in–hospital deaths from pulmonary embolism were 0 of 76 (0%) and all-cause mortality was 2 of 76 (2.6%). No septal motion abnormality was observed in 49 (64%), septal flattening in 25 (33%) and septal deviation in 2 (3%). None required ventilatory support, vasopressor therapy, rescue thrombolytic therapy, or catheter embolectomy. There were no in-hospital deaths due to pulmonary embolism. There was no difference in all-cause mortality comparing patients with and without right ventricular enlargement (relative risk = 1.04)
In-hospital prognosis is good in patients with pulmonary embolism and right ventricular enlargement if not in shock, acutely ill, on ventilatory support, recent myocardial infarction or life threatening arrhythmia. Right ventricular enlargement alone in patients with pulmonary embolism, therefore, does not appear to indicate a poor prognosis or an indication for thrombolytic therapy.
PMCID: PMC2219923  PMID: 18187071
Pulmonary embolism; venous thromboembolism; right ventricular dysfunction
21.  High-molecular-weight hyaluronan – a possible new treatment for sepsis-induced lung injury: a preclinical study in mechanically ventilated rats 
Critical Care  2008;12(4):R102.
Mechanical ventilation with even moderate-sized tidal volumes synergistically increases lung injury in sepsis and has been associated with proinflammatory low-molecular-weight hyaluronan production. High-molecular-weight hyaluronan (HMW HA), in contrast, has been found to be anti-inflammatory. We hypothesized that HMW HA would inhibit lung injury associated with sepsis and mechanical ventilation.
Sprague–Dawley rats were randomly divided into four groups: nonventilated control rats; mechanical ventilation plus lipopolysaccharide (LPS) infusion as a model of sepsis; mechanical ventilation plus LPS with HMW HA (1,600 kDa) pretreatment; and mechanical ventilation plus LPS with low-molecular-weight hyaluronan (35 kDa) pretreatment. Rats were mechanically ventilated with low (7 ml/kg) tidal volumes. LPS (1 or 3 mg/kg) or normal saline was infused 1 hour prior to mechanical ventilation. Animals received HMW HA or low-molecular-weight hyaluronan via the intraperitoneal route 18 hours prior to the study or received HMW HA (0.025%, 0.05% or 0.1%) intravenously 1 hour after injection of LPS. After 4 hours of ventilation, animals were sacrificed and the lung neutrophil and monocyte infiltration, the cytokine production, and the lung pathology score were measured.
LPS induced lung neutrophil infiltration, macrophage inflammatory protein-2 and TNFα mRNA and protein, which were decreased in the presence of both 1,600 kDa and 35 kDa hyaluronan pretreatment. Only 1,600 kDa hyaluronan completely blocked both monocyte and neutrophil infiltration and decreased the lung injury. When infused intravenously 1 hour after LPS, 1,600 kDa hyaluronan inhibited lung neutrophil infiltration, macrophage inflammatory protein-2 mRNA expression and lung injury in a dose-dependent manner. The beneficial effects of hyaluronan were partially dependent on the positive charge of the compound.
HMW HA may prove to be an effective treatment strategy for sepsis-induced lung injury with mechanical ventilation.
PMCID: PMC2575591  PMID: 18691420
22.  Atelectasis Induced by Thoracotomy Causes Lung Injury during Mechanical Ventilation in Endotoxemic Rats 
Journal of Korean Medical Science  2008;23(3):406-413.
Atelectasis can impair arterial oxygenation and decrease lung compliance. However, the effects of atelectasis on endotoxemic lungs during ventilation have not been well studied. We hypothesized that ventilation at low volumes below functional residual capacity (FRC) would accentuate lung injury in lipopolysaccharide (LPS)-pretreated rats. LPS-pretreated rats were ventilated with room air at 85 breaths/min for 2 hr at a tidal volume of 10 mL/kg with or without thoracotomy. Positive end-expiratory pressure (PEEP) was applied to restore FRC in the thoracotomy group. While LPS or thoracotomy alone did not cause significant injury, the combination of endotoxemia and thoracotomy caused significant hypoxemia and hypercapnia. The injury was observed along with a marked accumulation of inflammatory cells in the interstitium of the lungs, predominantly comprising neutrophils and mononuclear cells. Immunohistochemistry showed increased inducible nitric oxide synthase (iNOS) expression in mononuclear cells accumulated in the interstitium in the injury group. Pretreatment with PEEP or an iNOS inhibitor (1400 W) attenuated hypoxemia, hypercapnia, and the accumulation of inflammatory cells in the lung. In conclusion, the data suggest that atelectasis induced by thoracotomy causes lung injury during mechanical ventilation in endotoxemic rats through iNOS expression.
PMCID: PMC2526521  PMID: 18583875
Atelectasis; Functional Residual Capacity; Lung Injury; Nitric Oxide Synthase; Nitric Oxide Synthase Inhibitor
23.  Clinical Characteristics of Patients with Acute Pulmonary Embolism 
The American journal of medicine  2007;120(10):871-879.
Selection of patients for diagnostic tests for acute pulmonary embolism requires recognition of the possibility of pulmonary embolism based on the clinical characteristics. Patients in the Prospective Investigation of Pulmonary Embolism Diagnosis II (PIOPED II) had a broad spectrum of severity, which permits an evaluation of the subtle characteristics of mild pulmonary embolism as well as the characteristics of severe pulmonary embolism.
Data are from the national collaborative study, PIOPED II.
There may be dyspnea only on exertion. The onset of dyspnea is usually, but not always, rapid. Orthopnea may occur. In patients with pulmonary embolism in main or lobar pulmonary arteries, dyspnea or tachypnea occurred in 92%, but in only 65% in whom the largest pulmonary embolism was in segmental pulmonary arteries. In general, signs and symptoms were similar in elderly and younger patients, but dyspnea or tachypnea were less frequent in elderly patients with no prior cardiopulmonary disease. Dyspnea may be absent even in patients with circulatory collapse. Patients with a low probability objective clinical assessment sometimes had pulmonary embolism, even in proximal vessels.
Symptoms may be mild and generally recognized symptoms may be absent, particularly in patients with pulmonary embolism only in segmental pulmonary branches, but they may be absent even with severe pulmonary embolism. A high or intermediate probability objective clinical assessment may suggest the need for diagnostic studies, but a low probability objective clinical assessment does not exclude the diagnosis. Maintenance of a high level of suspicion is critical.
PMCID: PMC2071924  PMID: 17904458
Pulmonary embolism; venous thromboembolism; deep venous thrombosis; clinical diagnosis
24.  Suckling a protein-restricted rat dam leads to diminished albuminuria in her male offspring in adult life: a longitudinal study 
BMC Nephrology  2006;7:14.
Previous studies have shown that in male rats, exposure to maternal protein restriction either in utero or whilst suckling can have profound effects on both longevity and kidney telomere lengths. This study monitored albuminuria longitudinally in male rats whose mothers had been protein restricted either during pregnancy or lactation.
Pregnant Wistar rats were fed either a 20% ('control') or an 8% protein ('low protein') diet. At two days of age some of the pups were cross-fostered to dams fed the diet that was not given to their biological mothers. At weaning all pups were fed standard chow. Urine samples were collected for the measurement of albumin and creatinine at monthly intervals from two months-of-age. Longitudinal analysis was then performed using repeated measures analysis of variance.
Overall estimated marginal geometric mean (95 % confidence interval) urine albumin to creatinine ratios were: control animals 79.5 (57.2~110.6) g/mol (n = 6 litters, 24 animals in total), those exposed in utero to maternal protein restriction 71.0 (47.4~106.5) (n = 4 litters, 16 animals in total), those exposed to maternal protein restriction whilst suckling 21.2 (14.7~30.4) (n = 5 litters, 20 animals in total) (p < 0.001). These latter animals had lower albumin to creatinine ratios than either of the two other groups (both p < 0.001), which had ratios that were indistinguishable from each other (p = 1.0). Similar results were gained using 24 h. urine albumin excretion rates. These differences became evident from three months-of-age and were long-lasting.
Animals exposed to maternal protein restriction whilst suckling exhibited lower urine albumin excretions during much of adult life. As urine albumin can be nephrotoxic, these rats therefore appeared to be relatively protected against future nephron damage like that previously observed in animals exposed to maternal protein restriction in utero.
PMCID: PMC1592300  PMID: 17010194
25.  Role of Cytochrome P-450 in Alveolar Hypoxic Pulmonary Vasoconstriction in Dogs 
Journal of Clinical Investigation  1979;64(2):666-673.
Alveolar hypoxia induces pulmonary vasoconstriction by an unknown mechanism. Cytochrome P-450 (C-P450) is found in the lung and may modify pulmonary vascular tone via its sensitivity to changes in oxygen tension or by affecting metabolism of a chemical mediator. Metyrapone and carbon monoxide are both inhibitors of C-P450. We tested alveolar hypoxic pulmonary vasoconstriction (AHPV) in 20 dogs before, during, and after separate administration of each inhibitor. Anesthetized dogs were ventilated through a double lumen endotracheal tube allowing ventilation of one lung with N2 or CO as a hypoxic challenge and ventilation of the other lung with O2 to maintain adequate systemic oxygenation. Distribution of lung perfusion was determined with intravenous 133Xenon and external chest detectors. Before infusion of metyrapone, mean perfusion to the test lung decreased 30% with alveolar hypoxic challenge, but decreased only 10% during metyrapone infusion and returned to a base-line mean decrease of 31% after completion of metyrapone infusion. Prostaglandin F2 α and angiotensin II infusions produced equivalent increases in pulmonary vascular resistance before and during metyrapone infusion. Before CO, mean test lung perfusion decreased 31% with alveolar hypoxia but was reduced only 10% from control when unilateral end-tidal CO% was >75%. Washout of alveolar CO with unilateral N2 ventilation restored AHPV, with perfusion decreasing 29% from control. Thus, both metyrapone and carbon monoxide can reversibly inhibit AHPV. C-P450 may, therefore, be involved in the transduction process of the vasoconstrictor response to alveolar hypoxia.
PMCID: PMC372164  PMID: 457876

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