Chunling Fan

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) in chronic hypoxia- and antigen-mediated pulmonary vascular remodeling

BackgroundBoth chronic hypoxia and allergic inflammation induce vascular remodeling in the lung, but only chronic hypoxia appears to cause PH. We investigate the nature of the vascular remodeling and the expression and role of hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) in explaining this differential response.MethodsWe induced pulmonary vascular remodeling through either chronic hypoxia or antigen sensitization and challenge. Mice were evaluated for markers of PH and pulmonary vascular remodeling throughout the lung vascular bed as well as HIMF expression and genomic analysis of whole lung.ResultsChronic hypoxia increased both mean pulmonary artery pressure (mPAP) and right ventricular (RV) hypertrophy; these changes were associated with increased muscularization and thickening of small pulmonary vessels throughout the lung vascular bed. Allergic inflammation, by contrast, had minimal effect on mPAP and produced no RV hypertrophy. Only peribronchial vessels were significantly thickened, and vessels within the lung periphery did not become muscularized. Genomic analysis revealed that HIMF was the most consistently upregulated gene in the lungs following both chronic hypoxia and antigen challenge. HIMF was upregulated in the airway epithelial and inflammatory cells in both models, but only chronic hypoxia induced HIMF upregulation in vascular tissue.ConclusionsThe results show that pulmonary vascular remodeling in mice induced by chronic hypoxia or antigen challenge is associated with marked increases in HIMF expression. The lack of HIMF expression in the vasculature of the lung and no vascular remodeling in the peripheral resistance vessels of the lung is likely to account for the failure to develop PH in the allergic inflammation model.

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.

Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELMα) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature

Background Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo. Methodology/Principal Findings We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP)+ transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (∼20 µm) capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP+ BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP+ cells that localized to the pulmonary vasculature were α-smooth muscle actin+ and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner. Conclusions/Significance These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature.

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.

Choosing the right antibody for resistin-like molecule (RELM/FIZZ) family members

The family of resistin-like molecules (RELM), also known as found in inflammatory zone (FIZZ), consists of four members in mouse (RELMα/FIZZ1/HIMF, RELMβ/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in human (resistin and RELMβ). The importance of these proteins in many aspects of physiology and pathophysiology, especially inflammatory processes, is rapidly evolving in the literature, and many investigators are beginning to work in this field. Most published studies focus on only one isoform, do not evaluate other isoforms that might be present, and have not tested for the specificity of the antibody used. Because RELM isoforms have high sequence and structural similarity and both distinct and overlapping functions, it is important to use a specific antibody to distinguish each isoform in the study. We constructed and established HEK 293 cell lines that constitutively express each isoform. Using these cell lines, we determined the specificity of antibodies (both commercially available and laboratory-made) to each isoform by Western blot and immunofluorescence. Some of the antibodies showed specificity in Western blotting but were not applicable in immunofluorescence. Others showed cross reactivity in Western blot assays. Our results indicate that RELM antibody specificity should be taken into account when using them in research and interpreting data obtained with them.

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.