Background: Pulmonary arterial hypertension (PAH) is characterized by excessive remodeling of the distal arterioles and arteries, driven by endothelial cell (EC) apoptosis and uncontrolled mural cell proliferation. Increasing evidence suggests immune dysregulation in PAH, but one crucial part of the immune system, the pulmonary lymphatics, has been largely overlooked. Patients with idiopathic PAH (IPAH) often develop abnormal tertiary lymphoid structures adjacent to remodeled arteries, yet the role of lymphatics in PAH pathogenesis and vascular remodeling remains unclear.

Methods: Mice with lymphatic EC-specific fluorescent label (Prox1-CreERT2::Rosa26-LSL-tdT) or lymphatic EC-specific deletion of Vegfr3 (vascular endothelial growth factor receptor 3; Prox1-CreERT2::Vegfr3^fl/fl) were used. Pulmonary hypertension (PH) was induced in mice via exposure to hypoxia (10% FiO₂) for 1, 2, 3, or 6 weeks and in Sprague-Dawley rats via a single monocrotaline injection. MAZ51 was administered to inhibit Vegfr3. Right ventricular systolic pressure, right ventricular hypertrophy, and echocardiography were assessed. Immunofluorescent staining was performed in rodent and in IPAH lung sections for lymphatic EC analysis. Publicly available single-cell RNA sequencing data sets from human IPAH and mouse PH were reanalyzed to identify differentially expressed genes in lymphatic ECs.

Results: Pulmonary lymphatic vessels proliferated, branched, and dilated in response to hypoxia. Vegfr3 inhibition using MAZ51 or lymphatic EC-specific Vegfr3 deletion reduced hypoxia-induced lymphangiogenesis and was associated with worsened PH, right ventricular hypertrophy, and impaired drainage. Comparative single-cell RNA sequencing analysis revealed upregulation of CD74/Cd74 in lymphatic EC clusters from human IPAH and hypoxia-induced PH mouse lungs. In IPAH lymphatic ECs, increased CD74 expression was associated with increased FLT4 and activation of the VEGFR3-downstream MEK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) signaling pathways. Consistently, increased CD74 expression was found in lymphatic ECs from IPAH tissues, whereas CD74 overexpression in human lymphatic ECs enhanced VEGFR3 expression and was associated with impaired barrier permeability.

Conclusions: These findings identify adaptive lymphangiogenesis as a protective response in experimental PH and reveal a previously unrecognized CD74-VEGFR3 signaling axis in lymphatic ECs. Targeting lymphatic dysfunction may represent a novel therapeutic strategy to improve outcomes in PAH.

Ischemic stroke is a global health crisis necessitating targeted therapeutic strategies. Central to post-stroke pathology and repair is the CXCL12 signaling axis. In this review, we discuss the context-dependent roles of CXCL12 and its canonical receptor, CXCR4, within the post-ischemic microenvironment. The CXCL12/CXCR4 axis exhibits a temporal duality across the evolution of the neurovascular lesion; however, current evidence necessitates moving beyond a strictly binary framework. While the acute phase involves pathological cascades, such as blood-brain barrier disruption and leukocyte infiltration, the axis is simultaneously essential for recruiting protective innate immune subsets. During subsequent subacute and chronic phases, it governs essential restorative processes, including neurogenesis, angiogenesis, and remyelination. This complex temporal shift is mediated by the interplay between distinct CXCL12 isoforms and the regulatory influence of the atypical receptor ACKR3/CXCR7. Furthermore, these endogenous repair mechanisms exhibit synergies with non-pharmacological interventions, notably environmental enrichment and remote ischemic postconditioning. Our multidimensional model suggests that the functional outcome of CXCL12/CXCR4 signaling is determined by the intersection of timing, cell type, receptor availability, and adaptive responses to physiological stimuli. We synthesize fundamental mechanistic data with translational insights to evaluate the therapeutic potential of this axis and the pharmacological barriers to future regenerative strategies.

Duchenne Muscular Dystrophy (DMD) is caused by mutations in dystrophin, leading to degeneration and weakness of skeletal and cardiac muscle. Despite great progress in the development of gene replacement therapies, DMD remains a devastating disease. In our recent work, we demonstrated that loss of the cardiac Isl1-interacting protein (CIP), which interacts with dystrophin in sarcolemma of cardiomyocytes, accelerates the progression of dystrophic cardiomyopathy and identified Nox4 as one of the downstream mediators of this process. Here, we report that Setanaxib, a Nox1/4 inhibitor, protected the heart of CIP/Mdx double knockout (dKO) mice from heart failure; significantly, this compound also reduced cardiac fibrosis and protected against heart failure in Mdx/Utrn dKO mouse. At a molecular level, Nox1/4 inhibition reduced the expression of genes associated with cardiomyopathy in these animals. Further transcriptomic analysis of the hearts treated with Setanaxib revealed an enrichment in genes associated with fatty acid metabolism, oxidative phosphorylation, and protein binding, while genes related to epithelial-mesenchymal transition were downregulated. Collectively, these findings suggest that oxidative stress plays a key role in development of myocardial fibrosis and heart failure caused by dystrophin deficiency and suggest that Nox1/4 inhibitor treatment could be a novel therapy to treat DMD-associated cardiomyopathy.

Transcriptional reprogramming has an important role in kidney glomerular disease. Using in vivo murine models of podocyte injury, we studied the roles of the FOXC2 and WT1 transcription factors (TFs) in podocyte injury. Podocytes are a crucial cell type of glomeruli, the filtration units of each nephron. Podocyte injury is often the incipient event leading to chronic kidney disease. It is well established that the TFs FOXC2 and WT1 are required in podocytes to maintain the glomerular filtration barrier. Their role in the response to injury is less well understood. Here, we tested the hypothesis that FOXC2 and WT1 act together to mediate transcriptional reprogramming in response to podocyte injury. Similarly to that of WT1, genome-wide FOXC2 binding to target genes is dynamic during the course of injury, initially increasing, but late in injury there is a dramatic decrease in FOXC2 expression and in its binding to target genes. Podocyte-specific inactivation of FoxC2 or Wt1 in adult mice limits the transcriptional response to injury. Correlating FOXC2 and WT1 ChIP-seq analyses demonstrated that they co-bind many genes expressed in podocytes. Thus, reprogramming the transcriptome involves dynamic changes in the binding of FOXC2 and WT1 to their target genes during a reparative injury response.

Mutations in the AARS2 gene are linked to infantile cardiomyopathy; however, the underlying molecular mechanism remains unknown. Here, we report that PCBP1, a poly(rC) binding protein, interacts with the AARS2 transcript to mediate its alternative splicing. Cardiomyocyte-specific deletion of Pcbp1 in mice impairs normal splicing and causes premature termination of Aars2, leading to defects in heart development and postnatal lethality. Similarly, mice with a deletion in Aars2 that mimics a disease-causing splicing lesion display heart developmental abnormalities, reminiscent of those in patients with infantile mitochondrial cardiomyopathy. Mechanistically, loss of Pcbp1 or Aars2 in the heart reduces oxidative phosphorylation, a hallmark of patients with AARS2 mutations. This reduction in mitochondrial-encoded proteome activates mitonuclear communication and the UPR pathway, thereby inducing a compensatory nuclear-encoded mitochondrial gene program. Our findings provide insights into the PCBP1-AARS2 regulatory axis in mitochondrial cardiomyopathy.

Endocytic adaptor proteins are essential regulators of intracellular trafficking, coupling receptor internalization with downstream signaling. Recent discoveries have deepened our understanding of how endocytic adaptor proteins contribute to both physiological and pathological processes in the vascular system. This review summarizes the emerging roles of key adaptor proteins—such as epsins, disabled-2, and adaptor protein 2—across multiple vascular cell types, including endothelial cells, vascular smooth muscle cells, and macrophages. These adaptors have diverse but essential functions, ranging from regulating angiogenic signaling and maintaining endothelial barrier integrity to modulating inflammatory responses, vascular smooth muscle cells phenotypic switching, lipid uptake, and structural remodeling of blood vessels. We also highlight advances in therapeutic strategies, such as nanomedicine, viral expression vectors, DNA nanostructures, and small molecule inhibitors that target endocytic adaptors and their function. Understanding the diverse and cell-specific roles of endocytic adaptor proteins is important for gaining insights into vascular disease mechanisms and uncovering novel targets for therapeutic interventions. 

Microplastics (MPs) are small plastic particles emerging as significant environmental pollutants and humans are ubiquitously exposed to MPs. MPs can be detected in human atherosclerotic plaques and are associated with a higher risk of cardiovascular disease (CVD) and stroke in humans. However, the impact of MP exposure on the cardiovascular system remains elusive. In the current study, we investigated the effects of exposure to MPs at an environmentally relevant dose on atherosclerosis development in male and female low-density lipoprotein receptor-deficient (LDLR^-/-) mice. LDLR^-/- mice were fed a semisynthetic low-fat (4.3 %), low-cholesterol (0.02 %) diet and exposed to 10 mg/kg body weight MPs via daily oral gavage for 9 weeks. Male and female LDLR^-/- mice fed the low-fat diet did not develop obesity phenotype and exposure to MPs did not affect adiposity and circulating lipid profiles in those lean mice. Intriguingly, MP exposure increased atherosclerotic lesion areas in the aortic root by 63 % (p = 0.0185) and brachiocephalic artery by 624 % (p = 0.0541) in male LDLR^-/- mice but did not significantly affect atherosclerosis in female mice. Single-cell RNA sequencing analysis of the whole aorta revealed that exposure to MPs affected the proportions and cellular processes of key atherogenesis-related cell types, especially endothelia cells. Consistently, MP exposure induced pro-atherogenic gene expression in murine primary endothelia cells and human endothelial cells in vitro. Our findings reveal the sex-specific atherogenic effects of MPs in vivo and provide mechanistic insights and new understanding of the impact of MPs on atherosclerosis development and CVD risk in humans.

Background: Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-laden foam cells and plaques within the arterial wall. Dysfunctional vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages contribute to disease progression. Here, we report that macrophage-specific expression of epsins, highly conserved endocytic adaptor proteins involved in clathrin-mediated endocytosis, accelerates atherosclerosis in Western diet (WD)-fed mice.

Methods: Apoe-deficient (WT/Apoe^-/-) mice and littermates with a myeloid-specific deletion of epsin 1/2 on an Apoe^-/- background (LysM-DKO/Apoe^-/-) were generated and fed a WD for 16 weeks. Single-cell RNA sequencing (scRNA-seq) was conducted to investigate the cellular and molecular mechanisms regulated by macrophage epsins during atherosclerosis. Findings from scRNA-seq were validated through metabolic profiling, qRT-PCR, immunostaining, and co-culture experiments to assess associated phenotypic changes.

Results: LysM-DKO/Apoe^-/- mice exhibited significantly reduced atherosclerotic foam cell formation compared to WT/Apoe^-/- controls. scRNA-seq analysis identified 19 major cell types, including six VSMC and five macrophage subpopulations. Modulated VSMC1 and VSMC2 subtypes were associated with inflammation, migration, and VSMC-to-macrophage transition. These populations, along with foamy-Trem2 and inflammatory macrophages, were markedly reduced in LysM-DKO/Apoe^-/- mice. Transition of modulated VSMC2 subtype into macrophages was significantly inhibited, as confirmed by both computational analysis and experimental validation. Additionally, macrophage epsin deletion reversed endothelial dysfunction, suppressed cholesterol- and glucose-mediated signaling, and reduced expression of pro-inflammatory ligands IL-1β and TNF-⍺.

Conclusion: Macrophage epsin deletion limits foam cell formation and preserves VSMC and endothelial cell phenotypes and functions. These findings reveal a potential therapeutic strategy targeting macrophage epsins to combat atherosclerosis.