Single-Cell RNA Sequencing Reveals Repair Features of Human Umbilical Cord Mesenchymal Stromal Cells.

Rationale: The chronic lung disease bronchopulmonary dysplasia (BPD) is the most severe complication of extreme prematurity. BPD results in impaired lung alveolar and vascular development and long-term respiratory morbidity, for which only supportive therapies exist. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) improve lung structure and function in experimental BPD. Results of clinical trials with MSCs for many disorders do not yet match the promising preclinical studies. A lack of specific criteria to define functionally distinct MSCs persists. Objectives: To determine and correlate single-cell UC-MSC transcriptomic profiles with therapeutic potential. Methods: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs; control cells of mesenchymal origin) transcriptomes were investigated using single-cell RNA sequencing (scRNA-seq) analysis. The lung-protective effect of UC-MSCs with a distinct transcriptome and control HNDFs was tested in vivo in hyperoxia-induced neonatal lung injury in rats. Measurements and Main Results: UC-MSCs showed limited transcriptomic heterogeneity but were different from HNDFs. Gene Ontology enrichment analysis revealed distinct (progenitor-like and fibroblast-like) UC-MSC subpopulations. Only treatment with progenitor-like UC-MSCs improved lung function and structure and attenuated pulmonary hypertension in hyperoxia-exposed rat pups. Moreover, scRNA-seq identified major histocompatibility complex class I as a molecular marker of nontherapeutic cells and associated with decreased lung retention. Conclusions: UC-MSCs with a progenitor-like transcriptome, but not with a fibroblast-like transcriptome, provide lung protection in experimental BPD. High expression of major histocompatibility complex class I is associated with reduced therapeutic benefit. scRNA-seq may be useful to identify subsets of MSCs with superior repair capacity for clinical application.

Right Ventricular Maladaptation to Pressure Overload in Fischer Rats Is Associated With Profound Deficiency in Adenylate Kinase 1 and Impaired Ventricular Energetics.

BACKGROUND: We explored the mechanism of maladaptive right ventricular (RV) remodeling in Fischer compared with Sprague-Dawley (SD) rats exposed to pressure overload. METHODS: Pulmonary hypertension was induced by injection of the VEGFR antagonist, SU5416, followed by a 3-week exposure to hypoxia (Sugen chronic hypoxia). In vivo oxidative metabolism was assessed by RV/left ventricle ratio of [11C]acetate positron emission tomography clearance (kmono). Unbiased, global transcriptional and proteomic profiling was performed in Fischer and SD rats at baseline and after Sugen chronic hypoxia. RESULTS: All Fischer rats succumbed to RV failure by 5 weeks, whereas SD rats showed preserved RV function and 88% survival beyond 9 weeks (P<0.0001). Fischer rats exhibited increased oxidative metabolism at 4 weeks (P<0.05) and impaired RV efficiency compared with SD (work metabolic index: 52±10 versus 91±27 mmHg·mL/cm2, respectively; P<0.05), but no differences in mitochondrial complex activity. AK1 (adenylate kinase 1) was among the top 10 differentially expressed genes between Fischer and SD rats, with markedly lower RV expression in Fischer rats (FC: 3.36, P<0.05), confirmed by proteomic analysis and validated by Western blotting (>10-fold reduction, P<0.001). While whole-genome sequencing failed to reveal any coding region mutations in Fischer rats, there was a unique variant in a highly conserved upstream flanking region likely involved in the regulation of AK1 expression. CONCLUSIONS: Therefore, Fischer rats exhibit profound AK1 deficiency and inefficient cardiac energetics likely related to reduced adenosine triphosphate shuttling from the mitochondria to the contractile fibers. This represents a novel mechanism for RV failure in response to chronic increases in afterload.

Penetrance of Severe Pulmonary Arterial Hypertension in Response to Vascular Endothelial Growth Factor Receptor 2 Blockade in a Genetically Prone Rat Model Is Reduced by Female Sex.

Background We have previously reported important strain differences in response to SU5416 (SU, a vascular endothelial growth factor receptor 2 inhibitor) in rats and have identified a specific colony of Sprague-Dawley rats that are hyperresponsive (SDHR) to SU alone and develop severe pulmonary arterial hypertension (PAH) with a single injection of SU, even in the absence of hypoxia. Interestingly, SDHR rats exhibit incomplete penetrance of the severe PAH phenotype with an "all-or-none" response to SU alone, which provides a unique opportunity to assess the influence of female sex and sex hormones on susceptibility to PAH after endothelial injury in a genetically prone model. Methods and Results SDHR rats were injected with SU (20 mg/kg SC) and, in the absence of hypoxia, 72% of male but only 27% of female rats developed severe PAH at 7 weeks, which was associated with persistent endothelial cell apoptosis. This sex difference in susceptibility for severe PAH was abolished by ovariectomy. Estradiol replacement, beginning 2 days before SU (prevention), inhibited lung endothelial cell apoptosis and completely abrogated severe PAH phenotype in both male and ovariectomized female rats, while progesterone was only protective in ovariectomized female rats. In contrast, delayed treatment of SDHR rats with established PAH with estradiol or progesterone (initiated at 4 weeks post-SU) failed to reduce lung endothelial cell apoptosis or improve PAH phenotype. Conclusions Female sex hormones markedly reduced susceptibility for the severe PAH phenotype in response to SU alone in a hyperresponsive rat strain by abolishing SU-induced endothelial cell apoptosis, but did not reverse severe PAH in established disease.

Gene engineered mesenchymal stem cells: greater transgene expression and efficacy with minicircle vs. plasmid DNA vectors in a mouse model of acute lung injury.

BACKGROUND: Acute lung injury (ALI) and in its severe form, acute respiratory distress syndrome (ARDS), results in increased pulmonary vascular inflammation and permeability and is a major cause of mortality in many critically ill patients. Although cell-based therapies have shown promise in experimental ALI, strategies are needed to enhance the potency of mesenchymal stem cells (MSCs) to develop more effective treatments. Genetic modification of MSCs has been demonstrated to significantly improve the therapeutic benefits of these cells; however, the optimal vector for gene transfer is not clear. Given the acute nature of ARDS, transient transfection is desirable to avoid off-target effects of long-term transgene expression, as well as the potential adverse consequences of genomic integration. METHODS: Here, we explored whether a minicircle DNA (MC) vector containing human angiopoietin 1 (MC-ANGPT1) can provide a more effective platform for gene-enhanced MSC therapy of ALI/ARDS. RESULTS: At 24 h after transfection, nuclear-targeted electroporation using an MC-ANGPT1 vector resulted in a 3.7-fold greater increase in human ANGPT1 protein in MSC conditioned media compared to the use of a plasmid ANGPT1 (pANGPT1) vector (2048 ± 567 pg/mL vs. 552.1 ± 33.5 pg/mL). In the lipopolysaccharide (LPS)-induced ALI model, administration of pANGPT1 transfected MSCs significantly reduced bronchoalveolar lavage (BAL) neutrophil counts by 57%, while MC-ANGPT1 transfected MSCs reduced it by 71% (p < 0.001) by Holm-Sidak's multiple comparison test. Moreover, compared to pANGPT1, the MC-ANGPT1 transfected MSCs significantly reduced pulmonary inflammation, as observed in decreased levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2). pANGPT1-transfected MSCs significantly reduced BAL albumin levels by 71%, while MC-ANGPT1-transfected MSCs reduced it by 85%. CONCLUSIONS: Overall, using a minicircle vector, we demonstrated an efficient and sustained expression of the ANGPT1 transgene in MSCs and enhanced the therapeutic effect on the ALI model compared to plasmid. These results support the potential benefits of MC-ANGPT1 gene enhancement of MSC therapy to treat ARDS.

Efficacy of treprostinil in the SU5416-hypoxia model of severe pulmonary arterial hypertension: haemodynamic benefits are not associated with improvements in arterial remodelling.

BACKGROUND AND PURPOSE: Pulmonary arterial hypertension (PAH) is a life-threatening disease that leads to progressive pulmonary hypertension, right heart failure and death. Parenteral prostaglandins (PGs), including treprostinil, a prostacyclin analogue, represent the most effective medical treatment for severe PAH. We investigated the effect of treprostinil on established severe PAH and underlying mechanisms using the rat SU5416 (SU, a VEGF receptor-2 inhibitor)-chronic hypoxia (Hx) model of PAH. EXPERIMENTAL APPROACH: Male Sprague Dawley rats were injected with SU (20 mg·kg-1 , s.c.) followed by 3 weeks of Hx (10% O2 ) to induce severe PAH. Four weeks post-SU injection, baseline right ventricular (RV) systolic pressure (RVSP) was measured, and the rats were randomized to receive vehicle or treprostinil treatment (Trep-100: 100 ng·kg-1 ·min-1 or Trep-810: 810 ng·kg-1 ·min-1 ). Following 3 weeks of treatment, haemodynamic and echocardiographic assessments were performed, and tissue samples were collected for protein expression and histological analysis. KEY RESULTS: At week 7, no difference in RVSP or RV hypertrophy was observed between vehicle and Trep-100; however, Trep-810 significantly reduced RVSP and RV hypertrophy. Trep-810 treatment significantly improved cardiac structure and function. Further, a short-term infusion of treprostinil in rats with established PAH at 4 weeks post-SU produced an acute, dose-dependent reduction in RVSP consistent with a vasodilator effect. However, chronic Trep-810 treatment did not alter media wall thickness, degree of vascular occlusion or total vessel count in the lungs. CONCLUSIONS AND IMPLICATIONS: Treprostinil exerts therapeutic benefits in PAH through decreased vascular resistance and improved cardiac structure and function; however, treprostinil treatment does not have direct impact vascular remodelling.

Occlusive lung arterial lesions in endothelial-targeted, fas-induced apoptosis transgenic mice.

Pulmonary arterial hypertension (PAH) is a lethal disease that is characterized by functional and structural abnormalities involving distal pulmonary arterioles that result in increased pulmonary vascular resistance and ultimately right heart failure. In experimental models of pulmonary hypertension, endothelial cell (EC) apoptosis is a necessary trigger for the development of obliterative lung arteriopathy, inducing the emergence of hyperproliferative and apoptosis-resistant vascular cells. However, it has not been established whether EC apoptosis is sufficient for the induction of complex lung arteriolar lesions. We generated a conditional transgenic system in mice to test the hypothesis that lung endothelial cell apoptosis is sufficient to induce a PAH phenotype. The Fas-induced apoptosis (FIA) construct was expressed under the control of endothelial-specific Tie2 promoter (i.e., EFIA mice), and administration of a small molecule dimerizing agent, AP20187, resulted in modest pulmonary hypertension, which was associated with obliterative vascular lesions localized to distal lung arterioles in a proportion of transgenic mice. These lesions were characterized by proliferating cells, predominantly CD68 macrophages. Although endothelial cell apoptosis was also seen in the kidney, evidence of subsequent arteriopathy was seen only in the lung. This model provides direct evidence that lung endothelial cell apoptosis acts as a trigger to initiate a PAH phenotype and provides initial insight into the potential mechanisms that underlie a lung-specific arterial response to endothelial injury.

A lymphocyte-dependent mode of action for imatinib mesylate in experimental pulmonary hypertension.

The capacity of imatinib mesylate to reverse established pulmonary arterial hypertension (PAH) has been attributed to a reduction in pulmonary arterial muscularization via inhibition of platelet-derived growth factor receptor-ß on vascular smooth muscle cells. However, there is also a significant immunomodulatory component to the action of imatinib that may account for its efficacy in PAH. We found that monocrotaline-induced pulmonary hypertension was associated with a significant decrease in pulmonary natural killer (NK) cells and T lymphocytes and the accumulation of macrophages in the lungs of F344 rats. The prevention of pulmonary hypertension by imatinib blocked these changes in pulmonary leukocyte content and induced elevations in pulmonary interferon-γ, tumor necrosis factor α, and IL-10, corresponding to the enhanced activity of splenic NK cells ex vivo. Treatment with anti-asialo GM1 antiserum (ASGM1), which ablated circulating NK cells and depleted T cells, eliminated the therapeutic benefit of imatinib. ASGM1-treated animals also exhibited significant pulmonary arteriolar muscularization in response to monocrotaline challenge compared with immunocompetent controls despite daily imatinib administration to both groups. In the athymic rat, imatinib decreased right ventricular hypertrophy and pulmonary arteriolar muscularization in monocrotaline-challenged animals versus saline-treated controls but did not prevent pulmonary macrophage accumulation or the development of pulmonary hypertension. These data demonstrate that the immunomodulatory effects of imatinib are critical to its therapeutic action in experimental PAH.

Tie2-dependent neovascularization of the ischemic hindlimb is mediated by angiopoietin-2.

The angiopoietins (ANGPT) are ligands for the endothelial cell (EC) receptor tyrosine kinase, Tie2. Angpt-1 is a Tie2 agonist that promotes vascular maturation and stabilization, whereas Angpt-2 is a partial agonist/antagonist involved in the initiation of postnatal angiogenesis. Therefore, we hypothesized that overexpression of Angpt-2 would be more effective than Angpt-1 for enhancing the perfusion recovery in the ischemic hindlimb. Perfusion recovery was markedly impaired in Tie2-deficient animals at day 35 in a model of chronic hindlimb ischemia. Injections of Angpt-2 or VEGFA plasmid at 7 days post femoral artery resection enhanced recovery and improved arteriogenesis as assessed by angiographic scores, whereas Angpt-1 or null plasmid had no effect. In addition, Angpt-2 together with VEGF resulted in greater improvement in perfusion and collateral vessel formation than VEGF alone. Similarly, conditional overexpression of Angpt-2 in mice improved ischemic limb blood flow recovery, while Angpt-1 overexpression was ineffective. These data from Tie2 heterozygote deficient mice demonstrate, for the first time, the importance of the Tie2 pathway in spontaneous neovascularization in response to chronic hindlimb ischemia. Moreover, they show that overexpression of the partial agonist, Angpt-2, but not Angpt-1, enhanced ischemic hind limb perfusion recovery and collateralization, suggesting that a coordinated sequence antagonist and agonist activity is required for effective therapeutic revascularization.

Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis.

RATIONALE: Sepsis refers to the clinical syndrome of severe systemic inflammation precipitated by infection. Despite appropriate antimicrobial therapy, sepsis-related morbidity and mortality remain intractable problems in critically ill patients. Moreover, there is no specific treatment strategy for the syndrome of sepsis-induced multiple organ dysfunction. OBJECTIVES: We hypothesized that mesenchymal stem cells (MSCs), which have been shown to have immunomodulatory properties, would reduce sepsis-induced inflammation and improve survival in a polymicrobial model of sepsis. METHODS: Sepsis was induced in C57Bl/6J mice by cecal ligation and puncture (CLP), followed 6 hours later by an intravenous injection of MSCs or saline. Twenty-eight hours after CLP, plasma, bronchoalveolar lavage fluid and tissues were collected for analyses. Longer-term studies were performed with antibiotic coadministration to assess the effect of MSCs on survival. MEASUREMENTS AND MAIN RESULTS: MSC treatment significantly reduced mortality in septic mice receiving appropriate antimicrobial therapy. MSCs alone reduced systemic and pulmonary cytokine levels in mice with CLP-induced sepsis, preventing acute lung injury and organ dysfunction, despite the low levels of cell persistence. Microarray data highlighted an overall down-regulation of inflammation and inflammation-related genes (such as IL-10, IL-6) and a shift toward up-regulation of genes involved in promoting phagocytosis and bacterial killing. Finally, bacterial clearance was significantly greater in MSC-treated mice, in part due to enhanced phagocytotic activity of the host immune cells. CONCLUSIONS: These data demonstrate that MSCs have beneficial effects on experimental sepsis, possibly by paracrine mechanisms, and suggest that immunomodulatory cell therapy may be an effective adjunctive treatment to reduce sepsis-related morbidity and mortality.

The angiopietin-1-Tie2 pathway prevents rather than promotes pulmonary arterial hypertension in transgenic mice.

The role of the angiopoietin-1 (Ang1)-Tie2 pathway in the pathogenesis of pulmonary arterial hypertension (PAH) is controversial. Although Ang1 is well known to prevent endothelial activation and injury in systemic vascular beds, this pathway has been suggested to mediate pulmonary vascular remodeling in PAH. Therefore, we used transgenic models to determine the effect of increased or decreased Tie2 activity on the development of PAH. We now report modest spontaneous elevation in right ventricular systolic pressure in Tie2-deficient mice (Tie2(+/-)) compared with wild-type (WT) littermate controls, which was exacerbated upon chronic exposure to the clinically relevant PAH triggers, serotonin (5-HT) or interleukin-6 (IL-6). Moreover, overexpression of Ang1 in transgenic mice had no deleterious effect on pulmonary hemodynamics and, if anything, blunted the response to 5-HT. Exposure to 5-HT or IL-6 also decreased lung Ang1 expression, further reducing Tie2 activity and inducing pulmonary apoptosis in the Tie2(+/-) group only. Similarly, cultured pulmonary artery endothelial cells subjected to Tie2 silencing demonstrated increased susceptibility to apoptosis after 5-HT treatment. Finally, treatment of Tie2-deficient mice with Z-VAD, a pan-caspase inhibitor, prevented the pulmonary hypertensive response to 5-HT. Thus, these findings firmly establish that endothelial survival signaling via the Ang1-Tie2 pathway is protective in PAH.

Mice expressing BMPR2R899X transgene in smooth muscle develop pulmonary vascular lesions.

Familial pulmonary arterial hypertension (PAH) is associated with mutations in bone morphogenetic protein type II receptor (BMPR2). Many of these mutations occur in the BMPR2 tail domain, leaving the SMAD functions intact. To determine the in vivo consequences of BMPR2 tail domain mutation, we created a smooth muscle-specific doxycycline-inducible BMPR2 mutation with an arginine to termination mutation at amino acid 899. When these SM22-rtTA x TetO(7)-BMPR2(R899X) mice had transgene induced for 9 wk, starting at 4 wk of age, they universally developed pulmonary vascular pruning as assessed by fluorescent microangiography. Approximately one-third of the time, the induced animals developed elevated right ventricular systolic pressures (RVSP), associated with extensive pruning, muscularization of small pulmonary vessels, and development of large structural pulmonary vascular changes. These lesions included large numbers of macrophages and T cells in their adventitial compartment as well as CD133-positive cells in the lumen. Small vessels filled with CD45-positive and sometimes CD3-positive cells were a common feature in all SM22-rtTA x TetO(7)-BMPR2(R899X) mice. Gene array experiments show changes in stress response, muscle organization and function, proliferation, and apoptosis and developmental pathways before RVSP increases. Our results show that the primary phenotypic result of BMPR2 tail domain mutation in smooth muscle is pulmonary vascular pruning leading to elevated RVSP, associated with early dysregulation in multiple pathways with clear relevance to PAH. This model should be useful to the research community in examining early molecular and physical events in the development of PAH and as a platform to validate potential treatments.

Oxygen regulation of arterial smooth muscle cell proliferation and survival.

The purpose of this study was to determine if hypoxia elicits different proliferative and apoptotic responses in systemic arterial smooth muscle cells incubated under conditions that do or do not result in cellular ATP depletion and whether these effects are relevant to vascular remodeling in vivo. Gene expression profiling was used to identify potential regulatory pathways. In human aortic smooth muscle cells (HASMCs) incubated at 3% O(2), proliferation and progression through the G1/S interphase are enhanced. Incubation at 1% O(2) reduced proliferation, delayed G1/S transition, increased apoptotic cell death, and is associated with mitochondrial membrane depolarization and reduced cellular ATP levels. In aorta and mesenteric artery from rats exposed to hypoxia (10% O(2), 48 h), both proliferation and apoptosis are increased, as are medial nuclear density and smooth muscle cell content. Although nuclear levels of hypoxia-inducible factor 1-alpha (HIF-1alpha) are increased to a similar extent in HASMCs incubated at 1 and 3% O(2), expression of tumor protein p53, its transcriptional target p21, as well as their regulatory factors and downstream effectors, are differentially affected under these two conditions, suggesting that the bidirectional effects of hypoxia are mediated by this pathway. We conclude that hypoxia induces a state of enhanced cell turnover through increased rates of both smooth muscle cell proliferation and death. This confers the ability to remodel the vasculature in response to changing tissue metabolic needs while avoiding the accumulation of mutations that may lead to malignant transformation or the formation of abnormal vascular structures.

Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1.

BACKGROUND: The acute respiratory distress syndrome (ARDS), a clinical complication of severe acute lung injury (ALI) in humans, is a leading cause of morbidity and mortality in critically ill patients. ALI is characterized by disruption of the lung alveolar-capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Current specific treatment strategies for ALI/ARDS are lacking. We hypothesized that mesenchymal stem cells (MSCs), with or without transfection with the vasculoprotective gene angiopoietin 1 (ANGPT1) would have beneficial effects in experimental ALI in mice. METHODS AND FINDINGS: Syngeneic MSCs with or without transfection with plasmid containing the human ANGPT1 gene (pANGPT1) were delivered through the right jugular vein of mice 30 min after intratracheal instillation of lipopolysaccharide (LPS) to induce lung injury. Administration of MSCs significantly reduced LPS-induced pulmonary inflammation, as reflected by reductions in total cell and neutrophil counts in bronchoalveolar lavage (BAL) fluid (53%, 95% confidence interval [CI] 7%-101%; and 60%, CI 4%-116%, respectively) as well as reducing levels of proinflammatory cytokines in both BAL fluid and lung parenchymal homogenates. Furthermore, administration of MSCs transfected with pANGPT1 resulted in nearly complete reversal of LPS-induced increases in lung permeability as assessed by reductions in IgM and albumin levels in BAL (96%, CI 6%-185%; and 74%, CI 23%-126%, respectively). Fluorescently tagged MSCs were detected in the lung tissues by confocal microscopy and flow cytometry in both naïve and LPS-injured animals up to 3 d. CONCLUSIONS: Treatment with MSCs alone significantly reduced LPS-induced acute pulmonary inflammation in mice, while administration of pANGPT1-transfected MSCs resulted in a further improvement in both alveolar inflammation and permeability. These results suggest a potential role for cell-based ANGPT1 gene therapy to treat clinical ALI/ARDS.

Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease.

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance caused by narrowing and loss of pulmonary microvasculature, which in its late stages becomes refractory to traditional therapies. We hypothesized that bone marrow-derived endothelial progenitor cells (EPCs), which normally function to repair and regenerate blood vessels, would restore pulmonary hemodynamics and increase microvascular perfusion in the rat monocrotaline (MCT) model of PAH. Mononuclear cells were isolated from the bone marrow of syngeneic Fisher-344 rats by Ficoll gradient centrifugation and cultured for 7 to 10 days in endothelial growth medium. Fluorescently labeled endothelial-like progenitor cells (ELPCs) engrafted at the level of the distal pulmonary arterioles and incorporated into the endothelial lining in the MCT-injured lung. The administration of ELPCs 3 days after MCT nearly completely prevented the increase in right ventricular systolic pressure seen at 3 weeks with MCT alone (31.5+/-0.95 versus 48+/-3 mm Hg, respectively; P<0.001), whereas injection of skin fibroblasts had no protective effect (50.9+/-5.4 mm Hg). Delayed administration of progenitor cells 3 weeks after MCT prevented the further progression of PAH 2 weeks later (ie, 5 weeks after MCT), whereas only animals receiving ELPCs transduced with human endothelial NO-synthase (eNOS) exhibited significant reversal of established disease at day 35 (31+/-2 mm Hg, P<0.005) compared with day 21 (50+/-3 mm Hg). Fluorescent microangiography revealed widespread occlusion of pulmonary precapillary arterioles 3 weeks after MCT, whereas arteriolar-capillary continuity and microvascular architecture was preserved with the administration of syngeneic ELPCs. Moreover, the delivery of ELPCs to rats with established PAH resulted in marked improvement in survival, which was greatest in the group receiving eNOS-transduced cells. We conclude that bone marrow-derived ELPCs can engraft and repair the MCT-damaged lung, restoring microvasculature structure and function. Therefore, the regeneration of lung vascular endothelium by injection of progenitor cells may represent a novel treatment paradigm for patients with PAH.

WASp verprolin homology, cofilin homology, and acidic region domain-mediated actin polymerization is required for T cell development.

All members of the Wiskott-Aldrich syndrome protein (WASp) family contain a carboxyl-terminal verprolin homology, cofilin homology, and acidic region (VCA) domain that binds and activates the Arp2/3 complex, thereby linking these proteins to the induction of actin polymerization. Although the VCA domain imbues WASp and other WASp family members with the capacity to modulate cytoskeletal organization, little is known about the impact of this domain activity on lymphoid cell function. Here we demonstrate that T cell-restricted expression of VCA domain-deleted WASp (WASpdeltaVCA) in WAS(-/-) mice engenders a severe early block in T lymphopoiesis associated with impaired T cell antigen receptor alphabeta expression and a consequent failure to generate single-positive CD4(+) and CD8(+) T cells. These latter defects, which are not observed in WAS(-/-) mice, are associated with impaired induction of cellular actin polymerization and a failure in the terminal differentiation of double-negative thymocytes. These findings indicate that WASp family proteins play an essential role in modulating the signaling events required for early thymocyte development and reveal their capacity to subserve this role to depend on VCA domain-mediated actin polymerization.