Daniel J. Angelini

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) induces the vascular and hemodynamic changes of pulmonary hypertension

Pulmonary hypertension (PH) is a serious disease of multiple etiologies mediated by hypoxia, immune stimuli, and elevated pulmonary pressure that leads to vascular thickening and eventual right heart failure. In a chronic hypoxia model of PH, we previously reported the induction of a novel pleiotropic cytokine, hypoxia-induced mitogenic factor (HIMF), that exhibits mitogenic, vasculogenic, contractile, and chemokine properties during PH-associated vascular remodeling. To examine the role of HIMF in hypoxia-induced vascular remodeling, we performed in vivo knockdown of HIMF using short hairpin RNA directed at rat HIMF in the chronic hypoxia model of PH. Knockdown of HIMF partially blocked increases in mean pulmonary artery pressure, pulmonary vascular resistance, right heart hypertrophy, and vascular remodeling caused by chronic hypoxia. To demonstrate a direct role for HIMF in the mechanism of PH development, we performed HIMF-gene transfer into the lungs of rats using a HIMF-expressing adeno-associated virus (AAV). AAV-HIMF alone caused development of PH similar to that of chronic hypoxia with increased mean pulmonary artery pressure and pulmonary vascular resistance, right heart hypertrophy, and neomuscularization and thickening of small pulmonary arterioles. The findings suggest that HIMF represents a critical cytokine-like growth factor in the development of PH.

Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELMα) Increases Lung Inflammation and Activates Pulmonary Microvascular Endothelial Cells via an IL-4–Dependent Mechanism

Hypoxia-induced mitogenic factor (HIMF), also known as found in inflammatory zone 1 and resistin-like molecule α, belongs to a novel class of cysteine-rich secreted proteins. It exhibits mitogenic and chemotactic properties during pulmonary hypertension-associated vascular remodeling, as well as fibrogenic properties during pulmonary fibrosis. HIMF expression in the lung was reported to be regulated by Th2 cytokines (IL-4 and IL-13) via the transcription factor STAT6 pathway in a bleomycin-induced pulmonary fibrosis model. However, in this study, we found that in the hypoxia-induced pulmonary hypertension model, lung HIMF expression is increased in IL-4 and STAT6 knockout (KO) mice to the same degree as in wild-type (WT) mice, suggesting that induction of HIMF expression does not require Th2 regulation in this model. We also found that HIMF-induced proliferative activity, hypertrophy, collagen, and extracellular matrix deposition in the pulmonary arteries are significantly less in IL-4 KO mice than in WT mice. In addition, HIMF-induced production of angiogenic factors/chemokines, such as vascular endothelial growth factor, MCP-1, and stromal-derived factor-1, in the lung resident cells, as well as macrophage infiltration, were significantly suppressed in the lungs of IL-4 KO mice. We also show that IL-4 was significantly increased in the lungs of HIMF-treated WT mice. Our in vitro studies using pulmonary microvascular endothelial cells revealed that HIMF stimulated cell proliferation, vascular endothelial growth factor expression, and MCP-1 production in a manner that is dependent on the IL-4/IL-4Rα system. These findings suggest that IL-4 signaling may play a significant role in HIMF-induced lung inflammation and vascular remodeling.

Resistin-Like Molecule-β in Scleroderma-Associated Pulmonary Hypertension

Scleroderma is a systemic, mixed connective tissue disease that can impact the lungs through pulmonary fibrosis, vascular remodeling, and the development of pulmonary hypertension and right heart failure. Currently, little is known about the molecular mechanisms that drive this condition, but we have recently identified a novel gene product that is up-regulated in a murine model of hypoxia-induced pulmonary hypertension. This molecule, known as hypoxia-induced mitogenic factor (HIMF), is a member of the newly described resistin gene family. We have demonstrated that HIMF has mitogenic, angiogenic, vasoconstrictive, inflammatory, and chemokine-like properties, all of which are associated with vascular remodeling in the lung. Here, we demonstrate that the human homolog of HIMF, resistin-like molecule (RELM)-beta, is expressed in the lung tissue of patients with scleroderma-associated pulmonary hypertension and is up-regulated compared with normal control subjects. Immunofluorescence colocalization revealed that RELM-beta is expressed in the endothelium and vascular smooth muscle of remodeled vessels, as well as in plexiform lesions, macrophages, T cells, and myofibroblast-like cells. We also show that addition of recombinant RELM-beta induces proliferation and activation of ERK1/2 in primary cultured human pulmonary endothelial and smooth muscle cells. These results suggest that RELM-beta may be involved in the development of scleroderma-associated pulmonary hypertension.

IL-4 Is Proangiogenic in the Lung under Hypoxic Conditions

IL-4-mediated proangiogenic and proinflammatory vascular responses have been implicated in the pathogenesis of chronic lung diseases such as asthma. Although it is well known that hypoxia induces pulmonary angiogenesis and vascular alterations, the underlying mechanism of IL-4 on the pulmonary vasculature under hypoxic conditions remains unknown. In this context, we designed the present study to determine the functional importance of IL-4 for pulmonary angiogenesis under hypoxic conditions using IL-4 knockout (KO) animals. Our results show that hypoxia significantly increased IL-4Rα expression in wild-type (WT) control lungs. Even though hypoxia significantly up-regulated vascular endothelial growth factor (VEGF) receptor expression in the lungs of both genotypes, hypoxia-induced VEGF, VCAM-1, HIF-1α, and ERK phosphorylation were significantly diminished in IL-4 KO lungs as compared with WT control lungs. In addition, hypoxia-induced pulmonary angiogenesis and proliferating activities in the airway and pulmonary artery were significantly suppressed in IL-4 KO lungs as compared with WT control lungs. We also isolated primary lung fibroblasts from these genotypes and stimulated these cells with hypoxia. Hypoxia-induced VEGF production was significantly suppressed in lung fibroblasts from IL-4 KO mice. These in vitro results are in accordance with the in vivo data. Furthermore, we observed a significant increase of hypoxia-induced pulmonary angiogenesis in STAT6 KO mice similar to that in WT controls. In conclusion, IL-4 has proangiogenic properties in the lung under hypoxic conditions via the VEGF pathway, and this is independent of the STAT6 pathway.

S100A11 Mediates Hypoxia-induced Mitogenic Factor (HIMF)-induced Smooth Muscle Cell Migration, Vesicular Exocytosis, and Nuclear Activation

Hypoxia-induced mitogenic factor (HIMF) is a newly discovered protein that is up-regulated in murine models of pulmonary arterial hypertension and asthma. Our previous study shows that HIMF is a potent mitogenic, angiogenic, and vasoconstrictive chemokine associated with pulmonary arterial hypertension. Two-dimensional gel electrophoresis was used to investigate downstream molecules in HIMF-induced cell signaling, demonstrating that S100A11, an EF-hand calcium-binding protein, was exclusively altered and was decreased (2.7 ± 0.2-fold, p < 0.05) in pulmonary artery smooth muscle cells (SMCs) treated with HIMF for 5 min compared with untreated cells (n = 4). Immunofluorescence showed that in control cells S100A11 is a cytosolic protein, which then aggregates and translocates both to the plasma membrane with subsequent exocytosis and to the nucleus upon HIMF stimulation. Annexin A2, a known S100A11 binding partner, also colocalized with S100A11 during HIMF-induced membrane trafficking. To investigate the intracellular function of S100A11, siRNA was used to knock down S100A11 expression in SMCs. The S100A11 knockdown significantly reduced HIMF-induced SMC migration but did not affect the SMC mitogenic action of HIMF. Our data show that S100A11 mediates HIMF-induced smooth muscle cell migration, vesicular exocytosis, and nuclear activation.

Resistin-Like Molecule α Stimulates Proliferation of Mesenchymal Stem Cells While Maintaining Their Multipotency

Resistin-like molecule α (RELMα) is highly upregulated in the lungs of mice subjected to hypoxia. It is secreted from pulmonary epithelium and causes potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. By using bone marrow transplantation in mice, we previously showed that RELMα is able to increase the number of bone marrow-derived cells in lung tissue, especially in the remodeling pulmonary vasculature. The current study investigated the effect of RELMα on progenitor stem cell content in mouse lung. Hypoxia, while stimulating RELMα expression, caused an increase in the number of Sca1(+)/CD45(-) progenitor cells in lungs of wild-type mice, but not in lungs of RELMα knockout mice. An in vitro study with cultured mesenchymal stem cells (MSCs) showed that RELMα induced a robust proliferative response that was dependent on Phosphatidylinositol 3-kinase/Akt and Erk activation. RELMα treatment of MSCs caused upregulation of a large number of genes involved in cell cycle, mitosis, organelle, and cytoskeleton biogenesis, and DNA metabolism. MSCs cultured in RELMα-supplemented media were able to maintain their differentiation potential into adipogenic, osteogenic, or mesenchymal phenotypes, although adipogenic differentiation was partially inhibited. These results demonstrate that RELMα may be involved in stem cell proliferation in the lung, without affecting differentiation potential.

Resistin-Like Molecule α in Allergen-Induced Pulmonary Vascular Remodeling

Resistin-like molecule α (RELMα) has mitogenic, angiogenic, vasoconstrictive, and chemokine-like properties and is highly relevant in lung pathology. Here, we used RELMα knockout (Retnla(-/-)) mice to investigate the role of RELMα in pulmonary vascular remodeling after intermittent ovalbumin (OVA) challenge. We compared saline- and OVA-exposed wild-type (WT) mice and found that OVA induced significant increases in right ventricular systolic pressure, cardiac hypertrophy, pulmonary vascular remodeling of intra-alveolar arteries, goblet cell hyperplasia in airway epithelium, and intensive lung inflammation, especially perivascular inflammation. Genetic ablation of Retnla prevented the OVA-induced increase in pulmonary pressure and cardiac hypertrophy seen in WT mice. Histological analysis showed that Retnla(-/-) mice exhibited less vessel muscularization, less perivascular inflammation, reduced medial thickness of intra-alveolar vessels, and fewer goblet cells in upper airway epithelium (250-600 μm) than did WT animals after OVA challenge. Gene expression profiles showed that genes associated with vascular remodeling, including those related to muscle protein, contractile fibers, and actin cytoskeleton, were expressed at a lower level in OVA-challenged Retnla(-/-) mice than in similarly treated WT mice. In addition, bronchoalveolar lavage from OVA-challenged Retnla(-/-) mice had lower levels of cytokines, such as IL-1β, -1 receptor antagonist, and -16, chemokine (C-X-C motif) ligand 1, -2, -9, -10, and -13, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, TIMP metallopeptidase inhibitor-1, and triggering receptor expressed on myeloid cells-1, than did that from WT mice when analyzed by cytokine array dot blots. Retnla knockout inhibited the OVA-induced T helper 17 response but not the T helper 2 response. Altogether, our results suggest that RELMα is involved in immune response-induced pulmonary vascular remodeling and the associated increase in inflammation typically observed after OVA challenge.