Blood vessel endothelial cells (EC) display heterogeneity across vascular beds, which is anticipated to drive site-specific vascular pathology. This heterogeneity is assessed using transcriptomics in vivo, and functional assays in vitro, but how proteomes compare across human in vitro cultured ECs remains incompletely characterized. We generated an in-depth human EC proteomic landscape (>8000 proteins) across six organs and two in vitro models in steady-state and upon IFNγ-induced inflammation. EC proteomes displayed a high similarity and organ-specific proteins were limited. Variation between ECs was mainly based on proliferation and differentiation processes in which Blood outgrowth endothelial cells (BOEC) and Human umbilical vein cells (HUVEC) represented the extremes of proteomic phenotypes. The IFNγ response was highly conserved across all samples. Harnessing dynamics in protein abundances we delineated VWF and VE-Cadherin correlation networks. This EC landscape provides an extensive proteomic addition in studying EC biology and heterogeneity from an in vitro perspective.

Sphingolipids are eighteen carbon alcohol lipids synthesized from non-sphingolipid precursors in the endoplasmic reticulum (ER). The sphingolipids serve as precursors for a vast range of moieties found in our cells that play a critical role in various cellular processes, including cell division, senescence, migration, differentiation, apoptosis, pyroptosis, autophagy, nutrition intake, metabolism, and protein synthesis. In CVDs, different subclasses of sphingolipids and other derived molecules such as sphingomyelin (SM), ceramides (CERs), and sphingosine-1-phosphate (S1P) are directly related to diabetic cardiomyopathy, dilated cardiomyopathy, myocarditis, ischemic heart disease (IHD), hypertension, and atherogenesis. Several genome-wide association studies showed an association between genetic variations in sphingolipid pathway genes and the risk of CVDs. The sphingolipid pathway plays an important role in the biogenesis and secretion of exosomes. Small extracellular vesicles (sEVs)/ exosomes have recently been found as possible indicators for the onset of CVDs, linking various cellular signaling pathways that contribute to the disease progression. Important features of EVs like biocompatibility, and crossing of biological barriers can improve the pharmacokinetics of drugs and will be exploited to develop next-generation drug delivery systems. In this review, we have comprehensively discussed the role of sphingolipids, and sphingolipid metabolites in the development of CVDs. In addition, concise deliberations were laid to discuss the role of sEVs/exosomes in regulating the pathophysiological processes of CVDs and the exosomes as therapeutic targets.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients\' BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored.
METHODS: Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro.
RESULTS: Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model.
CONCLUSIONS: Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.

Cell therapy for adrenocortical insufficiency can potentially provide steroid replacement in response to physiological stimuli. Previously, we reported that adipose tissue-derived stromal cells (ADSCs) are transformed into steroid-producing cells by overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1). The steroidogenic cells are characterized by the production of both adrenal and gonadal steroids. Cytotherapy for adrenocortical insufficiency requires cells with more adrenocortical characteristics. Considering the highly developed vascular network within the adrenal cortex, all adrenocortical cells are adjacent to and interact with vascular endothelial cells (VECs). In this study, NR5A1-induced steroidogenic cells derived from mouse ADSCs (NR5A1-ADSCs) were co-cultured with mouse VECs. Testosterone secretion in NR5A1-ADSCs was not altered; however, corticosterone secretion significantly increased while levels of steroidogenic enzymes significantly increased in the corticosterone synthesis pathway. Co-culture with lymphatic endothelial cells (LECs) or ADSCs, or transwell culture with NR5A1-ADSCs and VECs did not alter corticosterone production. VECs expressed higher levels of collagen and laminin than LECs. Culture in type-IV collagen and laminin-coated dishes increased corticosterone secretion in NR5A1-ADSCs. These results suggest that VECs may characterize ADSC-derived steroidogenic cells into a more corticosterone-producing phenotype, and VECs may be useful for generating adrenal steroidogenic cells from stem cells.

BACKGROUND: In the repair of massive tissue defects using expanded large skin flaps, the incidence of complications increases with the size of the expanded area. Currently, stem cell therapy has limitations to solve this problem. We hypothesized that conditioned medium of adipose-derived stem cells (ADSC-CM) collected following mechanical pretreatment can assist skin expansion.
METHODS: Rat aortic endothelial cells and fibroblasts were cultured with ADSC-CM collected under 0%, 10%, 12%, and 15% stretching force. Ten-milliliter cylindrical soft tissue expanders were subcutaneously implanted into the backs of 36 Sprague-Dawley rats. The 0% and 10% stretch groups were injected with ADSC-CM collected under 0% and 10% stretching force, respectively, while the control group was not injected. After 3, 7, 14, and 30 days of expansion, expanded skin tissue was harvested for staining and qPCR analyses.
RESULTS: Endothelial cells had the best lumen formation and highest migration rate, and fibroblasts secreted the most collagen upon culture with ADSC-CM collected under 10% stretching force. The skin expansion rate was significantly increased in the 10% stretch group. After 7 days of expansion, the number of blood vessels in the expanded area, expression of the angiogenesis-associated proteins vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor, and collagen deposition were significantly increased in the 10% stretch group.
CONCLUSIONS: The optimal mechanical force upregulates specific paracrine proteins in ADSCs to increase angiogenesis and collagen secretion, and thereby promote skin regeneration and expansion. This study provides a new auxiliary method to expand large skin flaps.

Splenic tumors are very common in dogs, and canine hemangiosarcoma (HSA) is one of the most important malignant splenic tumors. Surgery followed by chemotherapy (anthracycline-based protocols) is recommended for treating canine HSA; however, patients still do not achieve long-term survival. Therefore, this research aimed to assess vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor-β (PDGFR-β) gene expression in formalin-fixed tissues, evaluate the quality of mRNA for quantitative polymerase chain reaction (qPCR) analysis and identify drug repositioning candidates based on VEGFR-2 and PDGFR-β. qPCR analysis identified the relative expression of heterogeneous VEGFR-2 and PDGFR-β, with samples showing no transcripts or very low expression and those with higher relative quantification for both genes. We then used immunohistochemistry to correlate the relative quantification of VEGFR-2 and PDGFR-β transcripts with respective higher protein expression to validate our results. In the next step, we evaluated drug repositioning candidates and identified small molecule inhibitors (i.e. sorafenib) and natural compounds (curcumin and resveratrol) with the ability to block VEGFR-2 and PDGFR-β genes. Overall, our results indicated that VEGFR-2 and PDGFR-β expression is highly variable among canine HSA samples and different drugs can block the expression of both genes. Therefore, a personalized approach could be useful for selecting anti-VEGFR-2 and PDGFR-β therapies and both genes are potential candidates for future oncological panels.
Os tumores esplênicos são muito comuns em cães, e o hemangiosarcoma (HSA) é um dos tumores esplênicos malignos mais importantes em cães. A cirurgia seguida de quimioterapia (protocolos baseados em antraciclinas) é a abordagem terapêutica mais recomendada para o tratamento da HSA canino; no entanto, os pacientes ainda não alcançam longa sobrevida após tratamento. Portanto, esta pesquisa teve como objetivo avaliar a expressão do receptor do fator de crescimento endotelial vascular-2 (VEGFR-2) e do receptor do fator de crescimento derivado de plaquetas-β (PDGFR-β) em tecidos fixados em formalina e identificar candidatos ao reposicionamento de medicamentos baseado na expressão desses genes. A análise qPCR identificou a expressão relativa heterogênea de VEGFR-2 e PDGFR-β, com amostras sem transcritos ou com expressão muito baixa ou amostras com alta quantificação relativa para ambos os genes. Em seguida, foi realizada o exame imuno-histoquímico para correlacionar a quantificação relativa dos transcritos de VEGFR-2 e PDGFR-β com a respectiva maior expressão proteica para validar nossos resultados. Na próxima etapa, avaliamos candidatos ao reposicionamento de medicamentos e identificamos inibidores de moléculas pequenas (ou seja, sorafenibe) e compostos naturais (curcumina e resveratrol) com capacidade de bloquear os genes VEGFR-2 e PDGFR-β. No geral, nossos resultados indicaram que a expressão de VEGFR-2 e PDGFR-β é altamente variável entre amostras caninas de HSA e diferentes drogas podem bloquear a expressão de ambos os genes. Portanto, uma abordagem personalizada poderia ser útil para selecionar terapias anti-VEGFR-2 e PDGFR-β e ambos os genes são potenciais candidatos para futuros painéis oncológicos.

Lymphatic vessels are essential for preventing the accumulation of harmful components within peripheral tissues, including the artery wall. Various endogenous mechanisms maintain adequate lymphatic function throughout life, with platelets being essential for preserving lymphatic vessel integrity. However, since lymph lacks platelets, their impact on the lymphatic system has long been viewed as restricted to areas where lymphatics intersect with blood vessels. Nevertheless, platelets can also exert long range effects through the release of extracellular vesicles (EVs) upon activation. We observed that platelet EVs (PEVs) are present in lymph, a compartment to which they could transfer regulatory effects of platelets. Here, we report that PEVs in lymph exhibit a distinct signature enabling them to interact with lymphatic endothelial cells (LECs). In vitro experiments show that the internalization of PEVs by LECs maintains their functional integrity. Treatment with PEVs improves lymphatic contraction capacity in atherosclerosis-prone mice. We suggest that boosting lymphatic pumping with exogenous PEVs offers a novel therapeutic approach for chronic inflammatory diseases characterized by defective lymphatics.

The endothelial glycocalyx (GCX), located on the luminal surface of vascular endothelial cells, is composed of glycoproteins, proteoglycans, and glycosaminoglycans. It plays a pivotal role in maintaining blood-brain barrier (BBB) integrity and vascular health within the central nervous system (CNS), influencing critical processes such as blood flow regulation, inflammation modulation, and vascular permeability. While the GCX is ubiquitously expressed on the surface of every cell in the body, the GCX at the BBB is highly specialized, with a distinct composition of glycans, physical structure, and surface charge when compared to GCX elsewhere in the body. There is evidence that the GCX at the BBB is disrupted and partially shed in many diseases that impact the CNS. Despite this, the GCX has yet to be a major focus of therapeutic targeting for CNS diseases. This review examines diverse model systems used in cerebrovascular GCX-related research, emphasizing the importance of selecting appropriate models to ensure clinical relevance and translational potential. This review aims to highlight the importance of the GCX in disease and how targeting the GCX at the BBB specifically may be an effective approach for brain specific targeting for therapeutics.

Kawasaki disease (KD) is a febrile illness characterised by systemic inflammation of small- and medium-sized blood vessels, which commonly occurs in young children. Although self-limiting, there is a risk of developing coronary artery lesions as the disease progresses, with delay in diagnosis and treatment. Unfortunately, the diagnosis of KD continues to remain a clinical dilemma. Thus, this article not only summarises the key research gaps associated with KD, but also evaluates the possibility of using circulating endothelial injury biomarkers, such as circulating endothelial cells, endothelial microparticles and vascular endothelial cell-free DNA, as diagnostic and prognostic tools for KD: a \"liquid biopsy\" approach. The challenges of translating liquid biopsies to use in KD and the opportunities for improvement in its diagnosis and management that such translation may provide are discussed. The use of endothelial damage markers, which are easily obtained via blood collection, as diagnostic tools is promising, and we hope this will be translated to clinical applications in the near future.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive increase in mean pulmonary arterial pressure. Mutations in the BMPR2 and AQP1 genes have been described in familial PAH. The bone morphogenetic proteins BMP9 and BMP10 bind with high affinity to BMPR2. Administration of BMP9 has been proposed as a potential therapeutic strategy against PAH, although recent conflicting evidence dispute the effect of such a practice. Considering the involvement of the above molecules in PAH onset, progression, and therapeutic value, we examined the effects of modulation of BMP9, BMPR2, and AQP1 on BMP9, BMP10, BMPR2, AQP1, and TGFB1 expression in human pulmonary microvascular endothelial cells in vitro. Our results demonstrated that silencing the BMPR2 gene resulted in increased expression of its two main ligands, namely BMP9 and BMP10. Exogenous administration of BMP9 caused the return of BMP10 to basal levels, while it restored the decreased AQP1 protein levels and the decreased TGFB1 mRNA and protein expression levels caused by BMPR2 silencing. Moreover, AQP1 gene silencing also resulted in increased expression of BMP9 and BMP10. Our results might possibly imply that the effect of exogenously administered BMP9 on molecules participating in the BMP signaling pathway could depend on the expression levels of BMPR2. Taken together, these results may provide insight into the highly complex interactions of the BMP signaling pathway.