Introduction and purpose Atherosclerosis is a chronic inflammatory disease of arteries, critically involving leukocytes like macrophages as well as T and B lymphocytes. Macrophages are major drivers of disease through the ingestion of lipoproteins, foam cell formation, and secretion of inflammatory mediators. Although macrophages outnumber other leukocytes in atherosclerotic plaques, T and B cells can shape the course of disease by promoting or mitigating inflammatory responses. Leukocytes highly express the phosphoinositide 3-kinase isoform delta (PI3Kd), exerting a key role in the regulation of immune responses including activation, proliferation, differentiation, and effector functions. Therefore, PI3Kd represents a promising target for the modulation of inflammatory diseases. Consequently, we aimed to analyse the role of PI3Kd in leukocytes during atherogenesis. Methods and results To investigate the role of PI3Kd in atherosclerosis, bone marrow from PI3Kd−/− or PI3Kd+/+ mice was transplanted into LDLR−/− mice. After a 6-weeks-challenge by high fat diet, PI3Kd−/− recipient LDLR−/− mice displayed profoundly impaired CD4+ and CD8+ T-cell numbers, CD4+ T-cell activation, CD4+ effector T-cell differentiation, and proatherogenic CD4+ T-helper (Th) 1 responses in para-aortic lymph nodes and spleen compared with PI3Kd+/+ transplanted controls. Surprisingly, the net effect of PI3Kd deficiency was a substantial increase of aortic inflammation and atherosclerosis in LDLR−/− mice. Whereas plaque content and functions of macrophages including foam cell formation, efferocytosis, and cytokine secretion remained unaffected, haematopoietic PI3Kd ablation strongly reduced mature B cells and serum immunoglobulins in LDLR−/− mice. Importantly, PI3Kd deficiency severely impaired numbers and immunosuppressive functions of regulatory CD4+ T cells (Tregs) in spleen, para-aortic lymph nodes, and plaques of LDLR−/− mice. Consequently, adoptive transfer of PI3Kd+/+ Tregs fully constrained the plaque burden in PI3Kd−/− transplanted LDLR−/− mice without affecting B-cell numbers and serum immunoglobulins, whereas adoptively transferred PI3Kd−/− Tregs were unable to relieve atherosclerosis progression. Conclusions Here, we demonstrate that PI3Kd plays a crucial role in Tregs, Th1 cells, and B cells during atherogenesis. Lack of PI3Kd signalling specifically in atheroprotective Treg responses outplays its impact on proatherogenic Th1 and B-cell responses, thus leading to aggravated atherosclerosis. Hence, PI3Kd is a key regulator of Treg biology and thereby protects against atherosclerosis. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Marga and Walter Boll Foundation
Introduction Pulmonary hypertension (PH) is a pulmonary vascular disease that is associated with unacceptably high morbidity and mortality. PH is characterized by chronically increased pulmonary arterial pressure, increased pulmonary vascular resistance and right ventricular (RV) dysfunction and hypertrophy. Underlying mechanisms include increased proliferation and reduced apoptosis of both vascular smooth muscle cells (SMC) and endothelial cells (EC), as well as dysregulated immune responses. We have previously shown that class IA phosphatidylinositol-3-kinase (PI3K) isoforms, activated via receptor tyrosine kinases, are critically involved in the pathogenesis of PH. However, recent findings suggest that the class IB isoform PI3Kγ, which is activated downstream of G protein coupled receptors, is also important. It has been shown that PI3Kγ is involved in numerous processes that promote both vascular remodelling and maladaptive cardiac hypertrophy, including leukocyte recruitment, expression of proinflammatory chemokines and cytokines, as well as SMC and EC proliferation and survival. Therefore, the aim of our study was to investigate the role of PI3Kγ in the pathogenesis of PH. Methods The impact of PI3Kγ on the pathogenesis of PH was analysed in vivo using mice expressing a catalytically inactive form of PI3Kγ (PI3KγKD/KD) in the hypoxia-induced mouse model of PH. Mice were kept at 10%O2 (HOX) for 21 days or left under normoxic conditions (NOX). Subsequently, systolic right ventricular pressure (RVSP) was measured with a pressure catheter. RV hypertrophy was expressed as the ratio of RV weight to left ventricular + septum weight. Migration and proliferation of human pulmonary arterial SMC (hPASMC) as well as EC (hMVEC) were analysed using a PI3Kγ isoform-specific inhibitor (AS605240 [0.1; 0.3; 1μM]). Chemotaxis was determined by means of a modified Boyden chamber, and proliferation was quantified by a Bromodeoxyuridine (BrdU) incorporation assay. Results Whereas PI3Kγ inactivation had no effect on NOX animals, hypoxia led to increased RVSP and RV hypertrophy in WT animals (34.67±2.02 mmHg; 0.38±0.087) which were unexpectedly further increased in PI3KγKD/KD mice (37.67±1.3 mmHg, p=0.0104 vs. HOX WT; 0.47±0.06, p=0.0155 vs. HOX WT). Heart rate and systemic blood pressure remained unchanged. Inhibition of PI3Kγ by means of AS605240 did not affect proliferation of hPASMC and hMVEC, induced by multiple stimuli (FCS [10%], PDGF-BB [30ng/ml], or CXCL12 [100ng/ml], VEGF [50ng/ml]), respectively. However, FCS-induced migration of these cells was significantly reduced by AS605240 [0.3μM] (p<0.05). Conclusion Contrary to our expectations, the results show that kinase inactivation of PI3Kγ was not able to attenuate the pathogenesis of PH, but surprisingly led to a significant increase without critically changing cellular responses of SMC and EC. Therefore, our results indicate an unexpected protective effect of PI3Kγ on PH. Funding Acknowledgement Type of funding source: None
Rationale Pulmonary arterial hypertension (PAH) is a vascular disease characterized by chronic increases in pulmonary vascular resistance (PVR), pulmonary arterial pressure (PAP), and right ventricular (RV) hypertrophy. Increased activation of receptor tyrosine kinase (RTK) -mediated signaling pathways leads to increased proliferation and migration of pulmonary smooth vascular muscle cells (PASMCs) which promote vascular remodeling processes. We identified the catalytic subunit p110alpha of phosphatidylinositol-3-kinase as a key enzyme for these processes and showed that both genetic ablation of p110alpha in SMCs and pharmacological inhibition can prevent experimental PH. Here, the effects of the orally bioavailable p110alpha selective PI3K inhibitor BYL719 on the RTK-mediated proliferation and chemotaxis of PASMCs, as well as the effects in the hypoxia-induced mouse and in the Sugen / hypoxia (SuHx) -induced rat model of PH were investigated. Methods Human and murine PASMCs were pretreated with different concentrations of BYL719 and stimulated with a mixture of growth factors (PDGF [30ng/ml], EGF [0,5ng/ml], bFGF [2ng/ml], insulin [0,5ng/ml], and FBS [5%]). Proliferation was investigated using a BrdU incorporation ELISA assay (Roche). Chemotaxis was quantified using modified Boyden chambers. Male BL/6 mice were subjected to hypoxia (10% O2) for 21 days. Treatment with BYL719 (or vehicle) was carried out via daily gavage of 50mg/kg bodyweight. In addition, a therapeutic approach was investigated using male Sprague Dawley rats in the SuHx model, which were treated with BYL719 (20 mg / kg body weight) or vehicle for 2 weeks after a three-week hypoxia phase. The RV pressure (RVSP) was measured using a Millar® or liquid-filled catheter. The RV hypertrophy is shown as the quotient of the weights of the RV to the LV + septum (RV / (LV + S)). Results Growth factor-induced proliferation and chemotaxis of the PASMCs were significantly and concentration-dependently inhibited by BYL719. The exposure to hypoxia led to an increase of the RVSP (24.5±0.95 to 35.2±1.28 mmHg) and the development of right ventricular hypertrophy (RV / LV + S 0.24±0.01 to 0.37±0.073), which was significantly reduced in the BYL719 treated group (RVSP 31.4±0.53 mmHg; RV / LV + S 0.31±0.01) (p<0.05). In addition, SuHx led to a robust increase of the RVSP (129.2±5.4 mmHg) and pronounced RV hypertrophy (RV / (LV + S): 0.86±0.04), which were significantly reduced by the therapeutic BYL719 treatment (102.0±6.1 mmHg or 0.64±0.03). Conclusion These results show that inhibition of p110alpha using the BYL719 reduced growth factor-mediated pathological processes in PASMCs in vitro, as well as hypoxia-induced (mouse) and already established SuHx-induced PH (rat). Thus, the inhibition of p110a using BYL719 represents a promising approach for the treatment of PAH. FUNDunding Acknowledgement Type of funding sources: None.
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