A potential adverse role for leptin and cardiac leptin receptor in the right ventricle in pulmonary arterial hypertension: effect of metformin is BMPR2 mutation-specific

IntroductionPulmonary arterial hypertension is a fatal cardiopulmonary disease. Leptin, a neuroendocrine hormone released by adipose tissue, has a complex relationship with cardiovascular diseases, including PAH. Leptin is thought to be an important factor linking metabolic syndrome and cardiovascular disorders. Given the published association between metabolic syndrome and RV dysfunction in PAH, we sought to determine the association between leptin and RV dysfunction. We hypothesized that in PAH-RV, leptin influences metabolic changes via leptin receptors, which can be manipulated by metformin.MethodsPlasma leptin was measured in PAH patients and healthy controls from a published trial of metformin in PAH. Leptin receptor localization was detected in RV from PAH patients, healthy controls, animal models of PH with RV dysfunction before and after metformin treatment, and cultured cardiomyocytes with two different BMPR2 mutants by performing immunohistochemical and cell fractionation studies. Functional studies were conducted in cultured cardiomyocytes to examine the role of leptin and metformin in lipid-driven mitochondrial respiration.ResultsIn human studies, we found that plasma leptin levels were higher in PAH patients and moderately correlated with higher BMI, but not in healthy controls. Circulating leptin levels were reduced by metformin treatment, and these findings were confirmed in an animal model of RV dysfunction. Leptin receptor expression was increased in PAH-RV cardiomyocytes. In animal models of RV dysfunction and cultured cardiomyocytes with BMPR2 mutation, we found increased expression and membrane localization of the leptin receptor. In cultured cardiomyocytes with BMPR2 mutation, leptin moderately influences palmitate uptake, possibly via CD36, in a mutation-specific manner. Furthermore, in cultured cardiomyocytes, the Seahorse XFe96 Extracellular Flux Analyzer and gene expression data indicate that leptin may not directly influence lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. However, metformin alone or when supplemented with leptin can improve lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. The effect of metformin on lipid-driven mitochondrial respiration in cardiomyocytes is BMPR2 mutation-specific.ConclusionIn PAH, increased circulating leptin can influence metabolic signaling in RV cardiomyocytes via the leptin receptor; in particular, it may alter lipid-dependent RV metabolism in combination with metformin in a mutation-specific manner and warrants further investigation

Overexpression of Msx1 in Mouse Lung Leads to Loss of Pulmonary Vessels Following Vascular Hypoxic Injury

Pulmonary arterial hypertension (PAH) is a progressive lung disease caused by thickening of the pulmonary arterial wall and luminal obliteration of the small peripheral arteries leading to increase in vascular resistance which elevates pulmonary artery pressure that eventually causes right heart failure and death. We have previously shown that transcription factor Msx1 (mainly expressed during embryogenesis) is strongly upregulated in transformed lymphocytes obtained from PAH patients, especially IPAH. Under pathological conditions, Msx1 overexpression can cause cell dedifferentiation or cell apoptosis. We hypothesized that Msx1 overexpression contributes to loss of small pulmonary vessels in PAH. In IPAH lung, MSX1 protein localization was strikingly increased in muscularized remodeled pulmonary vessels, whereas it was undetectable in control pulmonary arteries. We developed a transgenic mouse model overexpressing MSX1 (MSX1OE) by about 4-fold and exposed these mice to normoxic, sugen hypoxic (3 weeks) or hyperoxic (100% 02 for 3 weeks) conditions. Under normoxic conditions, compared to controls, MSX1OE mice demonstrated a 30-fold and 2-fold increase in lung Msx1 mRNA and protein expression, respectively. There was a significant retinal capillary dropout (p < 0.01) in MSX1OE mice, which was increased further (p < 0.03) with sugen hypoxia. At baseline, the number of pulmonary vessels in MSX1OE mice was similar to controls. In sugen-hypoxia-treated MSX1OE mice, the number of small (0–25 uM) and medium (25–50 uM) size muscularized vessels increased approximately 2-fold (p < 0.01) compared to baseline controls; however, they were strikingly lower (p < 0.001) in number than in sugen-hypoxia-treated control mice. In MSX1OE mouse lung, 104 genes were upregulated and 67 genes were downregulated compared to controls. Similarly, in PVECs, 156 genes were upregulated and 320 genes were downregulated from siRNA to MSX1OE, and in PVSMCs, 65 genes were upregulated and 321 genes were downregulated from siRNA to MSX1OE (with control in the middle). Many of the statistically significant GO groups associated with MSX1 expression in lung, PVECs, and PVSMCs were similar, and were involved in cell cycle, cytoskeletal and macromolecule organization, and programmed cell death. Overexpression of MSX1 suppresses many cell-cycle-related genes in PVSMCs but induces them in PVECs. In conclusion, overexpression of Msx1 leads to loss of pulmonary vessels, which is exacerbated by sugen hypoxia, and functional consequences of Msx1 overexpression are cell-dependent

BMP pathway regulation of and by macrophages.

Pulmonary arterial hypertension (PAH) is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC) treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle

NF-κB Activation Exacerbates, but Is not Required for Murine Bmpr2-Related Pulmonary Hypertension

Aim: The present study investigates the role of NF-κB in Bmpr2-related pulmonary hypertension (PH) using a murine model of PH with inducible overexpression of a cytoplasmic tail Bmpr2 mutation. Methods and Results: Electrophoretic mobility shift assay for nuclear extracts in Bmpr2R899X mouse lung and immunohistochemistry for NF-κB p65 in human PAH lung demonstrate that NF-κB is activated in end-stage disease. Acute inflammation or expression of a constitutively active NF-κB elicits a strong suppression of the BMP pathway in mice inversely correlating to activation of NF-κB targets. However, Bmpr2 mutation does not result in NF-κB activation in early disease development as assessed by luciferase reporter mice. Moreover, Bmpr2 mutant mice in which NF-κB activation is genetically blocked develop PH indistinguishable from that without the block. Finally, delivery of a virus causing NF-κB activation strongly exacerbates development of PH in Bmpr2 mutant mice, associated with increased remodeling. Conclusion: NF-κB activation exacerbates, but is not required for Bmpr2-related PH. Pulmonary vascular-specific activation of NF-κB may be a “second hit” that drives penetrance in heritable PH

Image_2_A potential adverse role for leptin and cardiac leptin receptor in the right ventricle in pulmonary arterial hypertension: effect of metformin is BMPR2 mutation-specific.JPEG

IntroductionPulmonary arterial hypertension is a fatal cardiopulmonary disease. Leptin, a neuroendocrine hormone released by adipose tissue, has a complex relationship with cardiovascular diseases, including PAH. Leptin is thought to be an important factor linking metabolic syndrome and cardiovascular disorders. Given the published association between metabolic syndrome and RV dysfunction in PAH, we sought to determine the association between leptin and RV dysfunction. We hypothesized that in PAH-RV, leptin influences metabolic changes via leptin receptors, which can be manipulated by metformin.MethodsPlasma leptin was measured in PAH patients and healthy controls from a published trial of metformin in PAH. Leptin receptor localization was detected in RV from PAH patients, healthy controls, animal models of PH with RV dysfunction before and after metformin treatment, and cultured cardiomyocytes with two different BMPR2 mutants by performing immunohistochemical and cell fractionation studies. Functional studies were conducted in cultured cardiomyocytes to examine the role of leptin and metformin in lipid-driven mitochondrial respiration.ResultsIn human studies, we found that plasma leptin levels were higher in PAH patients and moderately correlated with higher BMI, but not in healthy controls. Circulating leptin levels were reduced by metformin treatment, and these findings were confirmed in an animal model of RV dysfunction. Leptin receptor expression was increased in PAH-RV cardiomyocytes. In animal models of RV dysfunction and cultured cardiomyocytes with BMPR2 mutation, we found increased expression and membrane localization of the leptin receptor. In cultured cardiomyocytes with BMPR2 mutation, leptin moderately influences palmitate uptake, possibly via CD36, in a mutation-specific manner. Furthermore, in cultured cardiomyocytes, the Seahorse XFe96 Extracellular Flux Analyzer and gene expression data indicate that leptin may not directly influence lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. However, metformin alone or when supplemented with leptin can improve lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. The effect of metformin on lipid-driven mitochondrial respiration in cardiomyocytes is BMPR2 mutation-specific.ConclusionIn PAH, increased circulating leptin can influence metabolic signaling in RV cardiomyocytes via the leptin receptor; in particular, it may alter lipid-dependent RV metabolism in combination with metformin in a mutation-specific manner and warrants further investigation.</p

Primer sets.

<p>Primer sets.</p

Image_1_A potential adverse role for leptin and cardiac leptin receptor in the right ventricle in pulmonary arterial hypertension: effect of metformin is BMPR2 mutation-specific.JPEG

IntroductionPulmonary arterial hypertension is a fatal cardiopulmonary disease. Leptin, a neuroendocrine hormone released by adipose tissue, has a complex relationship with cardiovascular diseases, including PAH. Leptin is thought to be an important factor linking metabolic syndrome and cardiovascular disorders. Given the published association between metabolic syndrome and RV dysfunction in PAH, we sought to determine the association between leptin and RV dysfunction. We hypothesized that in PAH-RV, leptin influences metabolic changes via leptin receptors, which can be manipulated by metformin.MethodsPlasma leptin was measured in PAH patients and healthy controls from a published trial of metformin in PAH. Leptin receptor localization was detected in RV from PAH patients, healthy controls, animal models of PH with RV dysfunction before and after metformin treatment, and cultured cardiomyocytes with two different BMPR2 mutants by performing immunohistochemical and cell fractionation studies. Functional studies were conducted in cultured cardiomyocytes to examine the role of leptin and metformin in lipid-driven mitochondrial respiration.ResultsIn human studies, we found that plasma leptin levels were higher in PAH patients and moderately correlated with higher BMI, but not in healthy controls. Circulating leptin levels were reduced by metformin treatment, and these findings were confirmed in an animal model of RV dysfunction. Leptin receptor expression was increased in PAH-RV cardiomyocytes. In animal models of RV dysfunction and cultured cardiomyocytes with BMPR2 mutation, we found increased expression and membrane localization of the leptin receptor. In cultured cardiomyocytes with BMPR2 mutation, leptin moderately influences palmitate uptake, possibly via CD36, in a mutation-specific manner. Furthermore, in cultured cardiomyocytes, the Seahorse XFe96 Extracellular Flux Analyzer and gene expression data indicate that leptin may not directly influence lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. However, metformin alone or when supplemented with leptin can improve lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. The effect of metformin on lipid-driven mitochondrial respiration in cardiomyocytes is BMPR2 mutation-specific.ConclusionIn PAH, increased circulating leptin can influence metabolic signaling in RV cardiomyocytes via the leptin receptor; in particular, it may alter lipid-dependent RV metabolism in combination with metformin in a mutation-specific manner and warrants further investigation.</p

Gene expression alterations in bone marrow-derived macrophages (BMDM) from Bmpr2^delx4+ mice compared to controls.

<p>Shaded areas indicate SEM; all results are normalized to HPRT expression and then to control baseline. (<b>A</b>) Classical activation marker MRC1 is increased in BMDM from Bmpr2^delx4+ mice. (<b>B</b>) Alternative activation marker Itgam is decreased in BMDM from Bmpr2^delx4+ mice. (<b>C</b>) Canonical BMP pathway target Id1 is strongly suppressed by inflammation. (<b>D</b>) Secreted BMP pathway inhibitor Grem1 is induced by inflammation. (<b>E</b>) Intracellular BMP pathway inhibitor Smad6 is suppressed by inflammation. (<b>F</b>) In combination, these suggest that the BMP pathway normally suppresses classical macrophage activation; when macrophages are activated, they produce secreted factors to suppress the BMP pathway and intracellular factors to reduce suppression of the pathway.</p

The increased inflammatory cells in lungs from Bmpr2^delx4+ transgenic mice are primarily in the monocyte lineage.

<p>(<b>A</b>) Number of CD11b cells as a proportion of CD45+ leukocytes in the lung is increased in Rosa26-Bmpr2^delx4+ mice (p<.01 by ANOVA). (<b>B</b>) All categories of CD68 positive circulating cells showed significant increases (* = p<.05), with the increase numerically dominated by interstitial macrophages. (<b>C & D</b>) Some subtypes of alveolar macrophages and dendritic cells also show statistically significant increases (* = p<.05).</p