Qigong Exercise Balances Oxygen Supply and Acid-Base to Modulate Hypoxia: A Perspective Platform toward Preemptive Health & Medicine.

Qigong is a meditative movement with therapeutic effects and is commonly practiced in Eastern medicine. A growing body of evidence validates its health benefits, leading to mechanistic questions about how it works. We propose a novel mechanism by which the "acid" caused by hypoxia affects metabolism, and the way it is neutralized through Qigong practice involves the body's blood flow and vasculature modifications. Specifically, Qigong exercise generates an oxygen supply and acid-base balance against the hypoxic effects of underlying pathological conditions. We also propose that Qigong exercise mediated and focused on the local hypoxia environment of tissues might normalize the circulation of metabolic and inflammation accumulation in the tumor tissue and restore the normal metabolism of tissues and cells through calm, relaxation, and extreme Zen-style breathing that gravitates toward preemptive health and medicine. Thus, we propose the mechanisms of action related to Qigong, intending to unify Eastern and Western exercise theory.

Ectosome biogenesis and release processes observed by using live-cell dynamic imaging in mammalian glial cells.

BACKGROUND: Ectosomes are recognized as shedding from the plasma membranes into the extracellular environment. Recent research has demonstrated that ectosomes are surrounded by phospholipid membranes containing lipid rafts and caveolae. Some ectosomes contain cytokines in the lumen and have high levels of phosphatidylserine exposed to the outer membrane. Intracellular vesicles share both characters with ectosomes. Why the plasma membrane-derived ectosomes have the same characteristics as intracellular vesicles remain largely unknown. METHODS: Using live-cell dynamic imaging, we recorded the process of ectosome biogenesis and release in primary cultured neural cells. RESULTS: Our results show two different ectosome release methods: slow-releasing and fast-releasing. In the slow-releasing, multiple ectosomes emerge almost simultaneously on the cell surface and are released by outward budding from the plasma membrane. In the fast releasing, ectosomes squeeze out of the membrane domain and pinch off from a cell's surface. Using ER-tracker for live-cell imaging, we directly observed the process that intracellular vesicles jump out of the plasma membrane for release. This type of ectosomes has a reverse array of membrane proteins and phospholipids compared to the plasma membrane. So ectosomes should be divided into two groups: plasma membrane-derived and intracellular membrane-derived ectosomes. CONCLUSIONS: Both slow releasing and fast releasing EVs imply mechanisms of human diseases and for diagnostics and drug delivery.

Breathing Signature as Vitality Score Index Created by Exercises of Qigong: Implications of Artificial Intelligence Tools Used in Traditional Chinese Medicine.

Rising concerns about the short- and long-term detrimental consequences of administration of conventional pharmacopeia are fueling the search for alternative, complementary, personalized, and comprehensive approaches to human healthcare. Qigong, a form of Traditional Chinese Medicine, represents a viable alternative approach. Here, we started with the practical, philosophical, and psychological background of Ki (in Japanese) or Qi (in Chinese) and their relationship to Qigong theory and clinical application. Noting the drawbacks of the current state of Qigong clinic, herein we propose that to manage the unique aspects of the Eastern 'non-linearity' and 'holistic' approach, it needs to be integrated with the Western "linearity" "one-direction" approach. This is done through developing the concepts of "Qigong breathing signatures," which can define our life breathing patterns associated with diseases using machine learning technology. We predict that this can be achieved by establishing an artificial intelligence (AI)-Medicine training camp of databases, which will integrate Qigong-like breathing patterns with different pathologies unique to individuals. Such an integrated connection will allow the AI-Medicine algorithm to identify breathing patterns and guide medical intervention. This unique view of potentially connecting Eastern Medicine and Western Technology can further add a novel insight to our current understanding of both Western and Eastern medicine, thereby establishing a vitality score index (VSI) that can predict the outcomes of lifestyle behaviors and medical conditions.

A disposable fiber optic SPR probe for immunoassay.

Immunoassay can be divided into two aspects, one is immobilization of antibodies for the efficient detection of corresponding antigens, and the other is immobilization of antigens to search for antibodies which work against them. In this paper, we demonstrated these two aspects of immunoassay by using the disposable fiber optic biosensors based on surface plasmon resonance (SPR) through surface decoration with half-antibody fragments, which has been scarcely ever reported to the best of our knowledge. We first fabricated the fiber optic SPR biosensor which consists of one gold film coated single-mode fiber sandwiched by two multimode fibers. Then, we decorated the fiber optic SPR biosensors with antibody fragments and antigen fragments, respectively, and compared the specific detection performances of these two kinds of sensors. After surface decoration with half-antibody fragments, the antigen-decorated fiber probe has a demonstrated sensitivity and limit of detection of 0.9771nm/(µg/mL) and 0.1 µg/mL, respectively, which improves by 10 times compared with the performance of the antibody-decorated fiber probe. Additionally, the selective detection results indicate that our proposed biosensor can be employed as a reliable antigen detector or an effective antibody filter. Our proposed sensor has the advantages of miniaturization, low cost, simple usage, label-free detection, high efficiency and sensitivity, and can effectively avoid cross-contamination caused by reuse. Given the reliable and clean detection method for immunoassay, our work should open a new window for the utilization of miniaturized fiber optic sensors in biochemical sensing.

A label-free fiber optic SPR biosensor for specific detection of C-reactive protein.

A highly sensitive and label-free fiber optic surface plasmon resonance (SPR) biosensor for specific detection of C-reactive protein (CRP) is proposed and demonstrated. We take dopamine as a cross-linking agent to immobilize the anti-CRP monoclonal antibody, which is an efficient and simple method for specific modification of the fiber optic SPR sensor. The modified sensor can successfully detect CRP specifically. We realize the fabrication of a disposable fiber optic SPR sensor for the CRP specific detection. Through optimizing the immobilization time of anti-CRP monoclonal antibody and the reaction time of antigen and antibody experimentally, the sensor shows a satisfactory linear response (R2 = 0.97) to CRP concentration within the range from 0.01 to 20 µg/ml. Moreover, the highest CRP sensitivity is obtained at 1.17 nm per lg (µg/ml). With the advantages of simple structure and easy fabrication, our sensor is convenient to be batch produced and controlled with good consistency, which is especially suitable for the fabrication of disposable biosensor. It makes sense that our detection can effectively avoid the cross pollution caused by repeated use of the sensor.

Tissue elasticity regulated tumor gene expression: implication for diagnostic biomarkers of primitive neuroectodermal tumor.

BACKGROUND: The tumor microenvironment consists of both physical and chemical factors. Tissue elasticity is one physical factor contributing to the microenvironment of tumor cells. To test the importance of tissue elasticity in cell culture, primitive neuroectodermal tumor (PNET) stem cells were cultured on soft polyacrylamide (PAA) hydrogel plates that mimics the elasticity of brain tissue compared with PNET on standard polystyrene (PS) plates. We report the molecular profiles of PNET grown on either PAA or PS. METHODOLOGY/PRINCIPAL FINDINGS: A whole-genome microarray profile of transcriptional expression between the two culture conditions was performed as a way to probe effects of substrate on cell behavior in culture. The results showed more genes downregulated on PAA compared to PS. This led us to propose microRNA (miRNA) silencing as a potential mechanism for downregulation. Bioinformatic analysis predicted a greater number of miRNA binding sites from the 3' UTR of downregulated genes and identified as specific miRNA binding sites that were enriched when cells were grown on PAA-this supports the hypothesis that tissue elasticity plays a role in influencing miRNA expression. Thus, Dicer was examined to determine if miRNA processing was affected by tissue elasticity. Dicer genes were downregulated on PAA and had multiple predicted miRNA binding sites in its 3' UTR that matched the miRNA binding sites found enriched on PAA. Many differentially regulated genes were found to be present on PS but downregulated on PAA were mapped onto intron sequences. This suggests expression of alternative polyadenylation sites within intron regions that provide alternative 3' UTRs and alternative miRNA binding sites. This results in tissue specific transcriptional downregulation of mRNA in humans by miRNA. We propose a mechanism, driven by the physical characteristics of the microenvironment by which downregulation of genes occur. We found that tissue elasticity-mediated cytokines (TGFß2 and TNFα) signaling affect expression of ECM proteins. CONCLUSIONS: Our results suggest that tissue elasticity plays important roles in miRNA expression, which, in turn, regulate tumor growth or tumorigenicity.

Propidium iodide (PI) stains Nissl bodies and may serve as a quick marker for total neuronal cell count.

Propidium iodide (PI) reacts with both DNA and RNA and is a commonly used fluorescent reagent for nucleic acid staining. The aim of the study was to compare the cellular staining patterns of PI with that of Nissl staining in rat nervous tissues and to report a modified staining method that selectively labels Nissl bodies in neurons. Cryosections and paraffin sections of different tissues of normal Sprague-Dawley rats, including trigeminal ganglia, dorsal root ganglia, spinal cord, liver, and small intestine, were stained by either PI or the hematoxylin and eosin method. Some sections were treated with RNase or DNase before the above staining, and some were double stained with PI and a Nissl stain. The sections were observed by light, fluorescence or confocal microscopy. Results showed strong PI signals detected as patterns of granules in the neuronal cytoplasm of all nervous tissues, whereas the staining of neuronal nuclei was weaker. In contrast, nuclei of neuroglial cells were strongly stained by PI, while the cytoplasm was not obviously stained. Pretreatment of the neural tissue with RNase abolished the PI signals. Furthermore, the PI positive granules in neuronal cytoplasm co-localized with Nissl bodies stained by the fluorescent Nissl stain. When the tissue was pretreated with DNase, PI only stained the cytoplasmic granules of neurons, but not that of glial cells. Our results show that PI stains Nissl bodies and may serve as an economical and convenient neuron marker for neuronal cell counting when specific neural markers such as antibodies are not readily available.

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-1ß, -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.

KCNE2 modulates cardiac L-type Ca(2+) channel.

KCNE2 plays an important role in maintaining cardiac electrical stability. Mutations in KCNE2 have been linked to long-QT syndrome (LQT6) and atrial fibrillation/short QT syndrome. It has been suggested that KCNE2 has the most promiscuity of function which can interact with multiple-subunits of voltage-dependent cation channels and modulate their functions. However, whether KCNE2 regulates voltage-dependent L-type Ca(2)(+) channel (LCC) remains unknown. This study investigated the possible role of KCNE2 in regulating cardiac LCCs and the pathophysiological relevance of this regulation. We found that overexpression of KCNE2 in Sprague-Dawley rat cardiomyocytes decreased L-type Ca(2+)current (ICa,L), whereas KCNE2 knockdown by RNA interference increased ICa,L. Upregulation of KCNE2 caused a slight positive shift of the voltage-dependent activation and a negative shift of the steady-state voltage-dependent inactivation, and slowed the recovery from inactivation of ICa,L, while knockdown of KCNE2 had the contrary effects. Similar regulation of ICa,L magnitude had been observed in transfected HEK 293 cells. Coimmunoprecipitation and colocalization assays in both cardiomyocytes and the transfected cell line suggest that Cav1.2 physically interacted with KCNE2. Deletion of the N-terminal inhibitory module (NTI) of Cav1.2 results in the large loss of KCNE2 regulation of ICa,L and interaction with Cav1.2. Furthermore, we found that the familial atrial fibrillation related KCNE2 mutation R27C enhanced the effect of KCNE2 on suppressing ICa,L. Taken together, our findings indicate that KCNE2 modulates ICa,L by regulating NTI function of Cav1.2. The KCNE2 mutation R27C may induce familial atrial fibrillation partially through enhancing the suppression of ICa,L.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) in chronic hypoxia- and antigen-mediated pulmonary vascular remodeling.

BACKGROUND: Both 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. METHODS: We 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. RESULTS: Chronic 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. CONCLUSIONS: The 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.

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, RELMß/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in human (resistin and RELMß). 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.

The microtubule associated protein syntabulin is required for glucose-stimulated and cAMP-potentiated insulin secretion.

Syntabulin is a microtubule-associated protein that mediates anterograde transport of vesicles to neuronal processes. Here, we found that syntabulin was expressed in mouse pancreas and insulin-secreting ß-cells, and that it partially co-localized with microtubule and insulin-containing granules. The association of syntabulin with these organelles increased upon glucose stimulation. Knock-down of syntabulin by shRNA reduced both basal and glucose-stimulated insulin secretion, and diminished cAMP-Epac2 and cAMP-PKA potentiated insulin secretion. Additionally, syntabulin was preferentially phosphorylated by the Epac2 agonist 8-pCPT-2'-O-Me-cAMP, suggesting that syntabulin could be a novel effector of Epac2 and play a critical role in cAMP-enhanced insulin secretion.

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.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMalpha) 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.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) 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/RELM alpha) 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 (approximately 20 microm) 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 alpha-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.

Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP(3) pathway.

Hypoxia-induced mitogenic factor (HIMF), also known as "found in inflammatory zone 1" (FIZZ1) or resistin-like molecule-alpha (RELMalpha), is a profound vasoconstrictor of the pulmonary circulation and a strong mitogenic factor in pulmonary vascular smooth muscle. To further understand the mechanism of these contractile and mitogenic responses, we examined the effect of HIMF on intracellular Ca(2+) in human pulmonary artery smooth muscle cells (SMC). Ca(2+) imaging in fluo 4-loaded human pulmonary artery SMC revealed that recombinant murine HIMF increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in a sustained and oscillatory manner. This increase occurred independent of extracellular Ca(2+) influx. Pretreatment of human pulmonary artery SMC with U-73122, a specific inhibitor of phosphatidylinositol-phospholipase C (PLC) completely prevented the HIMF-induced Ca(2+) signal. The [Ca(2+)](i) increase was also abolished by pretreatment with 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist. Ryanodine pretreatment did not affect initiation of [Ca(2+)](i) activation or internal release but reduced [Ca(2+)](i) at the plateau phase. Pretreatment with the Galpha(i)-specific inhibitor pertussis toxin and the Galpha(s)-specific inhibitor NF-449 did not block the Ca(2+) signal. Knockdown of Galpha(q/11) expression did not prevent Ca(2+) release, but the pattern of Ca(2+) release changed from the sustained oscillatory transients with prolonged plateau to a series of short [Ca(2+)](i) transients that return to baseline. However, pretreatment with the tyrosine kinase inhibitor genistein completely inhibited the internal Ca(2+) release. These results demonstrate that HIMF can stimulate intracellular Ca(2+) release in human pulmonary artery SMC through the PLC signaling pathway in an IP(3)- and tyrosine phosphorylation-dependent manner and that Galpha(q/11) protein-coupled receptor and ryanodine receptor contribute to the increase of [Ca(2+)](i).

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-4R alpha 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-1alpha, 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.

Resistin-like molecule-beta 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.