The different natural estrogens promote endothelial healing through distinct cell targets.

The main estrogen, 17ß-estradiol (E2), exerts several beneficial vascular actions through estrogen receptor α (ERα) in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we report the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, which preserves smooth muscle cells (SMCs), E2, E3, and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4's action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its effects independently of ERα membrane-initiated signaling, in contrast with E2. Consistently, RNA sequencing analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differed from those of E2. Thus, whereas acceleration of endothelial healing by estrogens had been viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection.

Single-cell transcriptome mapping identifies a local, innate B cell population driving chronic rejection after lung transplantation.

Bronchiolitis obliterans syndrome (BOS) is the main reason for poor outcomes after lung transplantation (LTx). We and others have recently identified B cells as major contributors to BOS after LTx. The extent of B cell heterogeneity and the relative contributions of B cell subpopulations to BOS, however, remain unclear. Here, we provide a comprehensive analysis of cell population changes and their gene expression patterns during chronic rejection after orthotopic LTx in mice. Of 11 major cell types, Mzb1-expressing plasma cells (PCs) were the most prominently increased population in BOS lungs. These findings were validated in 2 different cohorts of human BOS after LTx. A Bhlhe41, Cxcr3, and Itgb1 triple-positive B cell subset, also expressing classical markers of the innate-like B-1 B cell population, served as the progenitor pool for Mzb1+ PCs. This subset accounted for the increase in IgG2c production within BOS lung grafts. A genetic lack of Igs decreased BOS severity after LTx. In summary, we provide a detailed analysis of cell population changes during BOS. IgG+ PCs and their progenitors - an innate B cell subpopulation - are the major source of local Ab production and a significant contributor to BOS after LTx.

Cathepsin B promotes collagen biosynthesis, which drives bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is a major complication after lung transplantation (LTx). BOS is characterised by massive peribronchial fibrosis, leading to air trapping-induced pulmonary dysfunction. Cathepsin B, a lysosomal cysteine protease, has been shown to enforce fibrotic pathways in several diseases. However, the relevance of cathepsin B in BOS progression has not yet been addressed. The aim of the study was to elucidate the function of cathepsin B in BOS pathogenesis.We determined cathepsin B levels in bronchoalveolar lavage fluid (BALF) and lung tissue from healthy donors (HD) and BOS LTx patients. Cathepsin B activity was assessed via a fluorescence resonance energy transfer-based assay and protein expression was determined using Western blotting, ELISA and immunostaining. To investigate the impact of cathepsin B in the pathophysiology of BOS, we used an in vivo orthotopic left LTx mouse model. Mechanistic studies were performed in vitro using macrophage and fibroblast cell lines.We found a significant increase of cathepsin B activity in BALF and lung tissue from BOS patients, as well as in our murine model of lymphocytic bronchiolitis. Moreover, cathepsin B activity was associated with increased biosynthesis of collagen and had a negative effect on lung function. We observed that cathepsin B was mainly expressed in macrophages that infiltrated areas characterised by a massive accumulation of collagen deposition. Mechanistically, macrophage-derived cathepsin B contributed to transforming growth factor-ß1-dependent activation of fibroblasts, and its inhibition reversed the phenotype.Infiltrating macrophages release active cathepsin B, thereby promoting fibroblast activation and subsequent collagen deposition, which drive BOS. Cathepsin B represents a promising therapeutic target to prevent the progression of BOS.

Smooth muscle cells-derived CXCL10 prevents endothelial healing through PI3Kγ-dependent T cells response.

AIMS: Defects in efficient endothelial healing have been associated with complication of atherosclerosis such as post-angioplasty neoatherosclerosis and plaque erosion leading to thrombus formation. However, current preventive strategies do not consider re-endothelialization in their design. Here, we investigate mechanisms linking immune processes and defect in re-endothelialization. We especially evaluate if targeting phosphoinositide 3-kinase γ immune processes could restore endothelial healing and identify immune mediators responsible for these defects. METHODS AND RESULTS: Using in vivo model of endovascular injury, we showed that both ubiquitous genetic inactivation of PI3Kγ and hematopoietic cell-specific PI3Kγ deletion improved re-endothelialization and that CD4+ T-cell population drives this effect. Accordingly, absence of PI3Kγ activity correlates with a decrease in local IFNγ secretion and its downstream interferon-inducible chemokine CXCL10. CXCL10 neutralization promoted re-endothelialization in vivo as the same level than those observed in absence of PI3Kγ suggesting a role of CXCL10 in re-endothelialization defect. Using a new established ex vivo model of carotid re-endothelialization, we showed that blocking CXCL10 restore the IFNγ-induced inhibition of endothelial healing and identify smooth muscle cells as the source of CXCL10 secretion in response to Th1 cytokine. CONCLUSION: Altogether, these findings expose an unforeseen cellular cross-talk within the arterial wall whereby a PI3Kγ-dependent T-cell response leads to CXCL10 production by smooth muscle cells which in turn inhibits endothelial healing. Therefore, both PI3Kγ and the IFNγ/CXCL10 axis provide novel strategies to promote endothelial healing.

Premedication with a cathepsin C inhibitor alleviates early primary graft dysfunction in mouse recipients after lung transplantation.

Neutrophil serine proteases (NSPs), like proteinase 3 (PR3) and neutrophil elastase (NE) are implicated in ischemia-reperfusion responses after lung transplantation (LTx). Cathepsin C (CatC) acts as the key regulator of NSP maturation during biosynthesis. We hypothesized that CatC inhibitors would reduce vascular breakdown and inflammation during reperfusion in pretreated lung transplant recipients by blocking NSP maturation in the bone marrow. An orthotopic LTx model in mice was used to mimic the induction of an ischemia-reperfusion response after 18 h cold storage of the graft and LTx. Recipient mice were treated subcutaneously with a chemical CatC inhibitor (ICatC) for 10 days prior to LTx. We examined the effect of the ICatC treatment by measuring the gas exchange function of the left lung graft, protein content, neutrophil numbers and NSP activities in the bone marrow 4 h after reperfusion. Pre-operative ICatC treatment of the recipient mice improved early graft function and lead to the disappearance of active NSP protein in the transplanted lung. NSP activities were also substantially reduced in bone marrow neutrophils. Preemptive NSP reduction by CatC inhibition may prove to be a viable and effective approach to reduce immediate ischemia reperfusion responses after LTx.

Inhibition of B cell-dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation.

Lung transplantation (LTx) is the only therapeutic option for many patients with chronic lung disease. However, long-term survival after LTx is severely compromised by chronic rejection (chronic lung allograft dysfunction [CLAD]), which affects 50% of recipients after 5 years. The underlying mechanisms for CLAD are poorly understood, largely due to a lack of clinically relevant animal models, but lymphocytic bronchiolitis is an early sign of CLAD. Here, we report that lymphocytic bronchiolitis occurs early in a long-term murine orthotopic LTx model, based on a single mismatch (grafts from HLA-A2:B6-knockin donors transplanted into B6 recipients). Lymphocytic bronchiolitis is followed by formation of B cell-dependent lymphoid follicles that induce adjacent bronchial epithelial cell dysfunction in a spatiotemporal fashion. B cell deficiency using recipient µMT-/- mice prevented intrapulmonary lymphoid follicle formation and lymphocytic bronchiolitis. Importantly, selective inhibition of the follicle-organizing receptor EBI2, using genetic deletion or pharmacologic inhibition, prevented functional and histological deterioration of mismatched lung grafts. In sum, we provided what we believe to be a mouse model of chronic rejection and lymphocytic bronchiolitis after LTx and identified intrapulmonary lymphoid follicle formation as a target for pharmacological intervention of long-term allograft dysfunction after LTx.

Preservation with α1-antitrypsin improves primary graft function of murine lung transplants.

BACKGROUND: Vascular damage and primary graft dysfunction increase with prolonged preservation times of transplanted donor lungs. Hence, storage and conservation of donated lungs in protein-free, dextran-containing electrolyte solutions, like Perfadex, is limited to about 6 hours. We hypothesized that transplanted lungs are protected against neutrophil-mediated proteolytic damage by adding α1-anti-trypsin (AAT), a highly abundant human plasma proteinase inhibitor, to Perfadex. METHODS: A realistic clinically oriented murine model of lung transplantation was used to simulate the ischemia-reperfusion process. Lung grafts were stored at 4°C in Perfadex solution supplemented with AAT or an AAT mutant devoid of elastase-inhibiting activity for 18 hours. We examined wild-type and proteinase 3/neutrophil elastase (PR3/NE) double-deficient mice as graft recipients. Gas exchange function and infiltrating neutrophils of the transplanted lung, as well as protein content and neutrophil numbers in the bronchoalveolar lavage fluid, were determined. RESULTS: AAT as a supplement to Perfadex reduced the extent of primary graft dysfunction and early neutrophil responses after extended storage for 18 hours at 4°C and 4-hour reperfusion in the recipients. Double-knockout recipients that lack elastase-like activities in neutrophils were also protected from early reperfusion injury, but not lung grafts that were perfused with a reactive center mutant of AAT devoid of elastase-inhibiting activity. CONCLUSIONS: PR3 and NE, the principal targets of AAT, are major triggers of post-ischemic reperfusion damage. Their effective inhibition in the graft and recipient is a promising strategy for organ usage after storage for >6 hours.

Cholesterol metabolism promotes B-cell positioning during immune pathogenesis of chronic obstructive pulmonary disease.

The development of chronic obstructive pulmonary disease (COPD) pathogenesis remains unclear, but emerging evidence supports a crucial role for inducible bronchus-associated lymphoid tissue (iBALT) in disease progression. Mechanisms underlying iBALT generation, particularly during chronic CS exposure, remain to be defined. Oxysterol metabolism of cholesterol is crucial to immune cell localization in secondary lymphoid tissue. Here, we demonstrate that oxysterols also critically regulate iBALT generation and the immune pathogenesis of COPD In both COPD patients and cigarette smoke (CS)-exposed mice, we identified significantly upregulated CH25H and CYP7B1 expression in airway epithelial cells, regulating CS-induced B-cell migration and iBALT formation. Mice deficient in CH25H or the oxysterol receptor EBI2 exhibited decreased iBALT and subsequent CS-induced emphysema. Further, inhibition of the oxysterol pathway using clotrimazole resolved iBALT formation and attenuated CS-induced emphysema in vivo therapeutically. Collectively, our studies are the first to mechanistically interrogate oxysterol-dependent iBALT formation in the pathogenesis of COPD, and identify a novel therapeutic target for the treatment of COPD and potentially other diseases driven by the generation of tertiary lymphoid organs.

The Activation Function-1 of Estrogen Receptor Alpha Prevents Arterial Neointima Development Through a Direct Effect on Smooth Muscle Cells.

RATIONALE: 17ß-Estradiol (E2) exerts numerous beneficial effects in vascular disease. It regulates gene transcription through nuclear estrogen receptor α (ERα) via 2 activation functions, AF1 and AF2, and can also activate membrane ERα. The role of E2 on the endothelium relies on membrane ERα activation, but the molecular mechanisms of its action on vascular smooth muscle cells (VSMCs) are not fully understood. OBJECTIVE: The aim of this study was to determine which cellular target and which ERα subfunction are involved in the preventive action of E2 on neointimal hyperplasia. METHODS AND RESULTS: To trigger neointimal hyperplasia of VSMC, we used a mouse model of femoral arterial injury. Cre-Lox models were used to distinguish between the endothelial- and the VSMC-specific actions of E2. The molecular mechanisms underlying the role of E2 were further characterized using both selective ERα agonists and transgenic mice in which the ERαAF1 function had been specifically invalidated. We found that (1) the selective inactivation of ERα in VSMC abrogates the neointimal hyperplasia protection induced by E2, whereas inactivation of endothelial and hematopoietic ERα has no effect; (2) the selective activation of membrane ERα does not prevent neointimal hyperplasia; and (3) ERαAF1 is necessary and sufficient to inhibit postinjury VSMC proliferation. CONCLUSIONS: Altogether, ERαAF1-mediated nuclear action is both necessary and sufficient to inhibit postinjury arterial VSMC proliferation, whereas membrane ERα largely regulates the endothelial functions of E2. This highlights the exquisite cell/tissue-specific actions of the ERα subfunctions and helps to delineate the spectrum of action of selective ER modulators.

Targeting PI3Kγ activity decreases vascular trauma-induced intimal hyperplasia through modulation of the Th1 response.

Interventional strategies to treat atherosclerosis, such as transluminal angioplasty and stent implantation, often cause vascular injury. This leads to intimal hyperplasia (IH) formation that induces inflammatory and fibroproliferative processes and ultimately restenosis. We show that phosphoinositide 3-kinase γ (PI3Kγ) is a key player in IH formation and is a valid therapeutic target in its prevention/treatment. PI3Kγ-deficient mice and mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. The transfer of PI3Kγ KD CD4(+) T cells into Rag2-deficient mice greatly reduced vascular occlusion compared with WT cells, clearly demonstrating the involvement of PI3Kγ in CD4(+) T cells during IH formation. In addition we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3Kγ activity, leading to decreased CXCL10 and RANTES production by smooth muscle cells. Finally, we show that treatment with the PI3Kγ inhibitor AS-605240 is sufficient to decrease IH in both mouse and rat models, reinforcing the therapeutic potential of PI3Kγ inhibition. Altogether, these findings demonstrate a new role for PI3Kγ activity in Th1-controlled IH development.