Aaron B. Waxman

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-&bgr; 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.

Exercise-Induced Pulmonary Arterial Hypertension

Background— The clinical relevance of exercise-induced pulmonary arterial hypertension (PAH) is uncertain, and its existence has never been well studied by direct measurements of central hemodynamics. Using invasive cardiopulmonary exercise testing, we hypothesized that exercise-induced PAH represents a symptomatic stage of PAH, physiologically intermediate between resting pulmonary arterial hypertension and normal. Methods and Results— A total of 406 consecutive clinically indicated cardiopulmonary exercise tests with radial and pulmonary arterial catheters and radionuclide ventriculographic scanning were analyzed. The invasive hemodynamic phenotype of exercise-induced PAH (n=78) was compared with resting PAH (n=15) and normals (n=16). Log-log plots of mean pulmonary artery pressure versus oxygen uptake (&OV0312;o2) were obtained, and a “join-point” for a least residual sum of squares for 2 straight-line segments (slopes m1, m2) was determined; m2m1=“takeoff” pattern. At maximum exercise, &OV0312;o2 (55.8±20.3% versus 66.5±16.3% versus 91.7±13.7% predicted) was lowest in resting PAH, intermediate in exercise-induced PAH, and highest in normals, whereas mean pulmonary artery pressure (48.4±11.1 versus 36.6±5.7 versus 27.4+3.7 mm Hg) and pulmonary vascular resistance (294±158 versus 161±60 versus 62±20 dyne · s · cm−5, respectively; P<0.05) followed an opposite pattern. An exercise-induced PAH plateau (n=32) was associated with lower &OV0312;o2max (60.6±15.1% versus 72.0±16.1% predicted) and maximum cardiac output (78.2±17.1% versus 87.8±18.3% predicted) and a higher resting pulmonary vascular resistance (247±101 versus 199±56 dyne · s · cm−5; P<0.05) than takeoff (n=40). The plateau pattern was most common in resting PAH, and the takeoff pattern was present in nearly all normals. Conclusions— Exercise-induced PAH is an early, mild, and clinically relevant phase of the PAH spectrum.

Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.

Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O2 on transgenic mice that overexpress IL-11 in the lung and transgene (-) controls. IL-11 markedly enhanced survival in 100% O2 with 100% of transgene (-) animals dying within 72-96 h and > 90% of transgene (+) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene (+) and (-) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene (+) as versus (-) mice after 100% O2 exposure, and IL-11 diminished hyperoxia-induced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation.

Ecteinascidin-743 (ET-743) for Chemotherapy-Naive Patients With Advanced Soft Tissue Sarcomas: Multicenter Phase II and Pharmacokinetic Study

PURPOSE To evaluate the response rate, toxicity profile, and pharmacokinetics of ecteinascidin-743 (ET-743) as first-line therapy in patients with unresectable advanced soft tissue sarcoma (STS). PATIENTS AND METHODS Thirty-six patients with STS were enrolled onto the study between September 1999 and August 2000. Patients were treated with 1.5 mg/m2 of ET-743 given as a 24-hour continuous intravenous (IV) infusion every 21 days. Pharmacokinetic sampling was performed in 23 patients. RESULTS One complete and five partial responses were achieved in 35 assessable patients for an overall response rate of 17.1% (95% CI, 6.6% to 33.6%). In addition, one patient had a minor response, leading to an overall clinical benefit of 20%. Neutropenia and transaminitis were the main grade 3 to 4 toxicities, which occurred in 33% and 36% of the patients. The estimated 1-year progression-free and overall survival rates were 21% (95% CI, 11% to 41%) and 72% (95% CI, 59% to 88%), respectively. Total body clearance (L/h) was not significantly correlated with body-surface area (r = -0.28; P = .21). Mild hepatic impairment or the extent of prior cytotoxic therapy does not seem to contribute significantly to the high interpatient variability (49%) in the clearance of this drug. Severity of treatment-related toxicity was not correlated with pharmacokinetic variables. CONCLUSION ET-743 demonstrates clinical activity as first-line therapy against STS with acceptable toxicity. Additional studies to establish empirical dosing guidelines may be necessary to improve the safety of the drug in patients with varying degrees of hepatic dysfunction and definitively establish the role of ET-743 for patients with these malignancies.

IL-13 stimulates vascular endothelial cell growth factor and protects against hyperoxic acute lung injury

Hyperoxia is an important cause of acute lung injury. To determine whether IL-13 is protective in hyperoxia, we compared the survival in 100% O(2) of transgenic mice that overexpress IL-13 in the lung and of nontransgenic littermate controls. IL-13 enhanced survival in 100% O(2). One hundred percent of nontransgenic mice died in 4-5 days, whereas 100% of IL-13-overexpressing mice lived for more than 7 days, and many lived 10-14 days. IL-13 also stimulated VEGF accumulation in mice breathing room air, and it interacted with 100% (2) to increase VEGF accumulation further. The 164-amino acid isoform was the major VEGF moiety in bronchoalveolar lavage from transgenic mice in room air, whereas the 120- and 188-amino acid isoforms accumulated in these mice during hyperoxia. In addition, antibody neutralization of VEGF decreased the survival of IL-13-overexpressing mice in 100% (2). These studies demonstrate that IL-13 has protective effects in hyperoxic acute lung injury. They also demonstrate that IL-13, alone and in combination with 100% (2), stimulates pulmonary VEGF accumulation, that this stimulation is isoform-specific, and that the protective effects of IL-13 are mediated, in part, by VEGF.

MicroRNA-21 Integrates Pathogenic Signaling to Control Pulmonary Hypertension Results of a Network Bioinformatics Approach

Background— Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA molecules are potential candidates for coordinated regulation of these disease stimuli. Methods and Results— Using a network biology approach, we identify microRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying microRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho-kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haploinsufficiency of BMP receptor type 2. To validate these predictions, we have found that hypoxia and BMP receptor type 2 signaling independently upregulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 downregulates BMP receptor type 2 expression. Furthermore, miR-21 directly represses RhoB expression and Rho-kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is upregulated in pulmonary tissue from several rodent models of PH and in humans with PH. On induction of disease in miR-21–null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH. Conclusions— A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying microRNA in PH.Background— Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA molecules are potential candidates for coordinated regulation of these disease stimuli. Methods and Results— Using a network biology approach, we identify microRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying microRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho-kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haploinsufficiency of BMP receptor type 2. To validate these predictions, we have found that hypoxia and BMP receptor type 2 signaling independently upregulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 downregulates BMP receptor type 2 expression. Furthermore, miR-21 directly represses RhoB expression and Rho-kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is upregulated in pulmonary tissue from several rodent models of PH and in humans with PH. On induction of disease in miR-21 –null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH. Conclusions— A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying microRNA in PH. # Clinical Perspective {#article-title-52}

Circulating Mitochondrial DNA in Patients in the ICU as a Marker of Mortality: Derivation and Validation

In this paper, Choi and colleagues analyzed levels of mitochondrial DNA in two prospective observational cohort studies and found that increased mtDNA levels are associated with ICU mortality, and improve risk prediction in medical ICU patients. The data suggests that mtDNA could serve as a viable plasma biomarker in MICU patients.

Systems-level regulation of microRNA networks by miR-130/301 promotes pulmonary hypertension

Development of the vascular disease pulmonary hypertension (PH) involves disparate molecular pathways that span multiple cell types. MicroRNAs (miRNAs) may coordinately regulate PH progression, but the integrative functions of miRNAs in this process have been challenging to define with conventional approaches. Here, analysis of the molecular network architecture specific to PH predicted that the miR-130/301 family is a master regulator of cellular proliferation in PH via regulation of subordinate miRNA pathways with unexpected connections to one another. In validation of this model, diseased pulmonary vessels and plasma from mammalian models and human PH subjects exhibited upregulation of miR-130/301 expression. Evaluation of pulmonary arterial endothelial cells and smooth muscle cells revealed that miR-130/301 targeted PPARγ with distinct consequences. In endothelial cells, miR-130/301 modulated apelin-miR-424/503-FGF2 signaling, while in smooth muscle cells, miR-130/301 modulated STAT3-miR-204 signaling to promote PH-associated phenotypes. In murine models, induction of miR-130/301 promoted pathogenic PH-associated effects, while miR-130/301 inhibition prevented PH pathogenesis. Together, these results provide insight into the systems-level regulation of miRNA-disease gene networks in PH with broad implications for miRNA-based therapeutics in this disease. Furthermore, these findings provide critical validation for the evolving application of network theory to the discovery of the miRNA-based origins of PH and other diseases.

Plasma gelsolin is a marker and therapeutic agent in animal sepsis

Objective:Plasma gelsolin is a circulating actin-binding protein that serves a protective role against tissue injuries. Depletion of plasma gelsolin in systemic inflammation may contribute to adverse outcomes. We examined the role of plasma gelsolin in animal models of sepsis. Design:Animal and laboratory experiments. Setting:Academic research laboratory. Subjects:Adult male mice. Interventions:Mice subjected to endotoxin or cecal ligation and puncture (CLP) were treated with exogenous plasma gelsolin or placebo. Measurements and Main Results:We document the depletion of plasma gelsolin (25–50% of normal) in murine models of sepsis associated with the presence of circulating actin within 6 hrs of septic challenge. Repletion of plasma gelsolin leads to solubilization of circulating actin aggregates and significantly reduces mortality in endotoxemic mice (survival rates were 88% in the gelsolin group vs. 0% in the saline group, p < .001) and in CLP-challenged mice (survival rates were 30% in the gelsolin group vs. 0% in the saline group, p = .001). Plasma gelsolin repletion also shifted the cytokine profile of endotoxemic mice toward anti-inflammatory (plasma interleukin-10 levels were 205 ± 108 pg/mL in the gelsolin group vs. 39 ± 29 pg/mL in the saline group, p = .02). Conclusions:We propose that circulation of particulate actin is a marker for sepsis-induced cell injury, that plasma gelsolin has a crucial protective role in sepsis, and that gelsolin replacement represents a potential therapy for this common lethal condition.

Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury

Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11-induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O(2) caused TUNEL(+) cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O(2). In A1-null mice, IL-11-induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11- and VEGF-induced cytoprotection.