Human Vascular Cells Research Articles

Hydrolytic instability of laser-ablatively deposited CaSi2 coatings in air and neutral water affects the behavior of bone healing-related cell types

Calcium silicide (CaSi2) instability in humid air or pH-neutral water remains unknown, although the topochemical formation of silicene from CaSi2 in various aqueous phases is a well-known process. Here we report on laser ablation of CaSi2 in the vacuum and ethanol, characterize coatings deposited on titanium substrates with electron microscopy, X-ray photoelectron, Raman, and infrared spectroscopy, and examine the instability of the deposited coatings in humid air and neutral water. We further investigate the behavior of human mesenchymal stromal cells (hMSCs), vascular cells (HUVECS, human umbilical vein endothelial cells), and macrophages (derived from THP-1 cell line) in contact with the deposited coatings submerged in cell culture medium. The observed results indicate that the CaSi2 coatings undergo topochemical conversion to silicene, which is accompanied by hydrolytic reactions leading to inorganic Ca compounds and SiO2, and that the response of all cell types in the hydrolyzed CaSi2 surface domain is negatively affected by the nature of the hydrolytic products. In contrast, cells showed a tendency for enhanced biocompatibility towards the CaSi2 particles ablated in the vacuum. The results suggest that coating approaches can significantly influence cell behavior outcomes.

Branched-chain α-ketoacids aerobically activate HIF1α signalling in vascular cells.

Hypoxia-inducible factor 1α (HIF1α) is a master regulator of biological processes in hypoxia. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in normal (primary) cells, remain elusive. Here we show that HIF1α signalling is activated in several human primary vascular cells in normoxia and in vascular smooth muscle cells of normal human lungs. Mechanistically, aerobic HIF1α activation is mediated by paracrine secretion of three branched-chain α-ketoacids (BCKAs), which suppress PHD2 activity via direct inhibition and via LDHA-mediated generation of L-2-hydroxyglutarate. BCKA-mediated HIF1α signalling activation stimulated glycolytic activity and governed a phenotypic switch of pulmonary artery smooth muscle cells, which correlated with BCKA metabolic dysregulation and pathophenotypic changes in pulmonary arterial hypertension patients and male rat models. We thus identify BCKAs as previously unrecognized signalling metabolites that aerobically activate HIF1α and that the BCKA-HIF1α pathway modulates vascular smooth muscle cell function, an effect that may be relevant to pulmonary vascular pathobiology.

Neddylation and Its Target Cullin 3 Are Essential for Adipocyte Differentiation.

The ongoing obesity epidemic has raised awareness of the complex physiology of adipose tissue. Abnormal adipocyte differentiation results in the development of systemic metabolic disorders such as insulin resistance and diabetes. The conjugation of NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to target protein, termed neddylation, has been shown to mediate adipogenesis. However, much remains unknown about its role in adipogenesis. Here, we demonstrated that neddylation and its targets, the cullin (CUL) family members, are differentially regulated during mouse and human adipogenesis. Inhibition of neddylation by MLN4924 significantly reduced adipogenesis of 3T3-L1 and human stromal vascular cells. Deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, suppressed neddylation and impaired adipogenesis. Neddylation deficiency did not affect mitotic cell expansion. Instead, it disrupted CREB/CEBPβ/PPARγ signaling, essential for adipogenesis. Interestingly, among the neddylation-targeted CUL family members, deletion of CUL3, but not CUL1, CUL2, or CUL4A, largely replicated the adipogenic defects observed with neddylation deficiency. A PPARγ agonist minimally rescued the adipogenic defects caused by the deletion of NAE1 and CUL3. In conclusion, our study demonstrates that neddylation and its targeted CUL3 are crucial for adipogenesis. These findings provide potential targets for therapeutic intervention in obesity and metabolic disorders.

Endothelial Cells Increase Mesenchymal Stem Cell Differentiation in Scaffold-Free 3D Vascular Tissue.

In this study, we present a versatile, scaffold-free approach to create ring-shaped engineered vascular tissue segments using human mesenchymal stem cell-derived smooth muscle cells (hMSC-SMCs) and endothelial cells (ECs). We hypothesized that incorporation of ECs would increase hMSC-SMC differentiation without compromising tissue ring strength or fusion to form tissue tubes. Undifferentiated hMSCs and ECs were co-seeded into custom ring-shaped agarose wells using four different concentrations of ECs: 0%, 10%, 20%, and 30%. Co-seeded EC and hMSC rings were cultured in SMC differentiation medium for a total of 22 days. Tissue rings were then harvested for histology, Western blotting, wire myography, and uniaxial tensile testing to examine their structural and functional properties. Differentiated hMSC tissue rings comprising 20% and 30% ECs exhibited significantly greater SMC contractile protein expression, endothelin-1 (ET-1)-meditated contraction, and force at failure compared with the 0% EC rings. On average, the 0%, 10%, 20%, and 30% EC rings exhibited a contractile force of 0.745 ± 0.117, 0.830 ± 0.358, 1.31 ± 0.353, and 1.67 ± 0.351 mN (mean ± standard deviation [SD]) in response to ET-1, respectively. Additionally, the mean maximum force at failure for the 0%, 10%, 20%, and 30% EC rings was 88.5 ± 36. , 121 ± 59.1, 147 ± 43.1, and 206 ± 0.8 mN (mean ± SD), respectively. Based on these results, 30% EC rings were fused together to form tissue-engineered blood vessels (TEBVs) and compared with 0% EC TEBV controls. The addition of 30% ECs in TEBVs did not affect ring fusion but did result in significantly greater SMC protein expression (calponin and smoothelin). In summary, co-seeding hMSCs with ECs to form tissue rings resulted in greater contraction, strength, and hMSC-SMC differentiation compared with hMSCs alone and indicates a method to create a functional 3D human vascular cell coculture model.

Zibotentan in Microvascular Angina: A Randomized, Placebo-Controlled, Crossover Trial.

Background: Microvascular angina is associated with dysregulation of the endothelin system and impairments in myocardial blood flow, exercise capacity, and health-related quality of life. The G allele of the noncoding single nucleotide polymorphism RS9349379 enhances expression of the endothelin-1 gene (EDN1) in human vascular cells, potentially increasing circulating concentrations of Endothelin-1 (ET-1). Whether zibotentan, an oral ET-A receptor selective antagonist, is efficacious and safe for the treatment of microvascular angina is unknown. Methods: Patients with microvascular angina were enrolled in this double-blind, placebo-controlled, sequential crossover trial of zibotentan (10 mg daily for 12 weeks). The trial population was enriched to ensure a G allele frequency of 50% for the RS9349379 single nucleotide polymorphism. Participants and investigators were blinded to genotype. The primary outcome was treadmill exercise duration (seconds) using the Bruce protocol. The primary analysis estimated the mean within-participant difference in exercise duration after treatment with zibotentan versus placebo. Results: A total of 118 participants (mean ±SD; years of age 63.5 [9.2 ]; 71 [60.2% ] females; 25 [21.2% ] with diabetes) were randomized. Among 103 participants with complete data, the mean exercise duration with zibotentan treatment compared with placebo was not different (between-treatment difference, -4.26 seconds [95 ] CI, -19.60 to 11.06] P=0.5871). Secondary outcomes showed no improvement with zibotentan. Zibotentan reduced blood pressure and increased plasma concentrations of ET-1. Adverse events were more common with zibotentan (60.2%) compared with placebo (14.4%; P<0.001). Conclusions: Among patients with microvascular angina, short-term treatment with a relatively high dose (10 mg daily) of zibotentan was not beneficial. Target-related adverse effects were common.

3D Brain Vascular Niche Model Captures Invasive Behavior and Gene Signatures of Glioblastoma.

Glioblastoma (GBM) is a lethal brain cancer with no effective treatment; understanding how GBM cells respond to tumor microenvironment remains challenging as conventional cell cultures lack proper cytoarchitecture while in vivo animal models present complexity all at once. Developing a culture system to bridge the gap is thus crucial. Here, we employed a multicellular approach using human glia and vascular cells to optimize a 3-dimensional (3D) brain vascular niche model that enabled not only long-term culture of patient derived GBM cells but also recapitulation of key features of GBM heterogeneity, in particular invasion behavior and vascular association. Comparative transcriptomics of identical patient derived GBM cells in 3D and in vivo xenotransplants models revealed that glia-vascular contact induced genes concerning neural/glia development, synaptic regulation, as well as immune suppression. This gene signature displayed region specific enrichment in the leading edge and microvascular proliferation zones in human GBM and predicted poor prognosis. Gene variance analysis also uncovered histone demethylation and xylosyltransferase activity as main themes for gene adaption of GBM cells in vivo . Furthermore, our 3D model also demonstrated the capacity to provide a quiescence and a protective niche against chemotherapy. In summary, an advanced 3D brain vascular model can bridge the gap between 2D cultures and in vivo models in capturing key features of GBM heterogeneity and unveil previously unrecognized influence of glia-vascular contact for transcriptional adaption in GBM cells featuring neural/synaptic interaction and immunosuppression.

Circ_0008571 modulates the phenotype of vascular smooth muscle cells by targeting miR-145-5p in intracranial aneurysms

BackgroundThe dysfunction of human vascular smooth cells (hVSMCs) is significantly connected to the development of intracranial aneurysms (IAs). By suppressing the activity of microRNAs (miRNAs), circular RNAs (circRNAs) participate in IA pathogenesis. Nevertheless, the role of hsa_circ_0008571 in IAs remains unclear. MethodscircRNA sequencing was used to identify circRNAs from human IA tissues. To determine the function of circ_0008571, Transwell, wound healing, and cell proliferation assays were conducted. To identify the target of circ_0008571, the analyses of CircInteractome and TargetScan, as well as the luciferase assay were carried out. Furthermore, circ_0008571 knockdown and over-expression were performed to investigate its functions in IA development and the underlying molecular mechanisms. ResultsBoth hsa_circ_0008571 and Integrin beta 8 (ITGB8) were downregulated, while miR-145-5p transcription was elevated in the aneurysm wall of IAs patients compared to superficial temporal artery tissues. In vitro, cell migration and growth were dramatically suppressed after hsa_circ_0008571 overexpression. Mechanistically, has_circ_0008571 could suppress miR-145-5p activity by direct sponging. Moreover, we found that ITGB8 expression and the activation of the TGF-β-mediated signaling pathway were significantly enhanced. ConclusionThe hsa_circ_0008571-miR-145-5p-ITGB8 axis plays an essential role in IA progression.

Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells.

Hypoxia-inducible factor 1α (HIF1α) is a master regulator of numerous biological processes under low oxygen tensions. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in primary cells remain elusive. Here, we show that HIF1α signaling is activated in several human primary vascular cells under ambient oxygen tensions, and in vascular smooth muscle cells (VSMCs) of normal human lung tissue, which contributed to a relative resistance to further enhancement of glycolytic activity in hypoxia. Mechanistically, aerobic HIFα activation is mediated by paracrine secretion of three branched chain α-ketoacids (BCKAs), which suppress prolyl hydroxylase domain-containing protein 2 (PHD2) activity via direct inhibition and via lactate dehydrogenase A (LDHA)-mediated generation of L-2-hydroxyglutarate (L2HG). Metabolic dysfunction induced by BCKAs was observed in the lungs of rats with pulmonary arterial hypertension (PAH) and in pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH patients. BCKA supplementation stimulated glycolytic activity and promoted a phenotypic switch to the synthetic phenotype in PASMCs of normal and PAH subjects. In summary, we identify BCKAs as novel signaling metabolites that activate HIF1α signaling in normoxia and that the BCKA-HIF1α pathway modulates VSMC function and may be relevant to pulmonary vascular pathobiology.

MiR-92b-3p modulates apoptosis in smooth muscle cells through BCL2L11 regulation, unravelling novel therapeutic strategies for unstable plaques

Abstract Funding Acknowledgements None. Smooth muscle cell (SMC) apoptosis is a contributory factor to plaque rupture in atherosclerosis, thereby promoting plaque destabilization through various mechanisms such as thrombogenic activation and plaque microcalcification. Immunohistochemical analysis has revealed elevated expression of proteins associated with intrinsic apoptosis, such as BCL2L11 (Bim), in unstable atherosclerotic plaques. Our hypothesis posits that Bim is a direct target of miR-92b-3p, an understudied microRNA, prompting an investigation into its role in regulating apoptosis in SMCs. Our objective is to discern a novel therapeutic axis for preventing plaque rupture by employing miRNA-based targeted interventions. Within a model of primary human vascular cells, we employed various in vitro techniques, including PCR, Western Blot, Live cell imaging, and ELISA. Lipotransfection facilitated transient modulation of miR-92b-3p, using both knockdown and elevation, while an siRNA transfection model was also utilized. MiR-92b-3p exhibits robust expression in late atherosclerotic lesions in vivo (p&amp;lt;0.001) and demonstrates heightened levels in smooth muscle cells (SMCs) compared to endothelial cells (ECs) in vitro (p&amp;lt;0.01) and in vivo in respective tissues (p&amp;lt;0.01). Bim, a direct target of miR-92b-3p, experiences upregulation post-miR-92b-3p knockdown and downregulation following pre-miR-92b-3p introduction into SMCs, both at mRNA and protein levels (p&amp;lt;0.01, p&amp;lt;0.01). Apoptosis increases in growth-medium cultured SMCs after miR-92b-3p knockdown (p&amp;lt;0.05), but not in senescent SMCs, ECs, or basal-medium treated SMCs. Live-cell imaging reveals a reduction in SMC count after anti-miR-92b-3p transfection (p&amp;lt;0.01), accompanied by cell shrinkage indicative of apoptosis 72 hours post-transfection (p&amp;lt;0.05). Growth factor stimulation results in miR-92b-3p upregulation (p&amp;gt;0.05) and Bim downregulation in SMCs. SiRNA knockdown of BIM in SMCs abolishes apoptosis induction through miR-92b-3p knockdown (p&amp;lt;0.05). Conversely, in apoptotic SMCs, miR-92b-3p upregulation protects cells from apoptosis (p&amp;lt;0.05). MiR-92b-3p effectively modulates mitochondrial apoptosis in SMCs by targeting BIM. This regulatory impact is exclusive to proliferative SMCs, known for their deleterious involvement in atherosclerotic plaques. Cumulatively, the elevation of miR-92b-3p via targeted miRNA therapeutics or miRNA-drug interactions in the advanced stages of atherosclerosis presents a potential therapeutic avenue for averting plaque rupture.

Development and characterization of lipid nanocapsules loaded with iron oxide nanoparticles for magnetic targeting to the blood–brain barrier

Brain drug delivery is severely hindered by the presence of the blood–brain barrier (BBB). Its functionality relies on the interactions of the brain endothelial cells with additional cellular constituents, including pericytes, astrocytes, neurons, or microglia. To boost brain drug delivery, nanomedicines have been designed to exploit distinct delivery strategies, including magnetically driven nanocarriers as a form of external physical targeting to the BBB. Herein, a lipid-based magnetic nanocarrier prepared by a low-energy method is first described. Magnetic nanocapsules with a hydrodynamic diameter of 256.7 ± 8.5 nm (polydispersity index: 0.089 ± 0.034) and a ξ-potential of -30.4 ± 0.3 mV were obtained. Transmission electron microscopy-energy dispersive X-ray spectroscopy analysis revealed efficient encapsulation of iron oxide nanoparticles within the oily core of the nanocapsules. Both thermogravimetric analysis and phenanthroline-based colorimetric assay showed that the iron oxide percentage in the final formulation was 12 wt.%, in agreement with vibrating sample magnetometry analysis, as the specific saturation magnetization of the magnetic nanocapsules was 12% that of the bare iron oxide nanoparticles. Magnetic nanocapsules were non-toxic in the range of 50–300 μg/mL over 72 h against both the human cerebral endothelial hCMEC/D3 and Human Brain Vascular Pericytes cell lines. Interestingly, higher uptake of magnetic nanocapsules in both cell types was evidenced in the presence of an external magnetic field than in the absence of it after 24 h. This increase in nanocapsules uptake was also evidenced in pericytes after only 3 h. Altogether, these results highlight the potential for magnetic targeting to the BBB of our formulation.Graphical

Protein interaction networks in the vasculature prioritize genes and pathways underlying coronary artery disease

Population-based association studies have identified many genetic risk loci for coronary artery disease (CAD), but it is often unclear how genes within these loci are linked to CAD. Here, we perform interaction proteomics for 11 CAD-risk genes to map their protein-protein interactions (PPIs) in human vascular cells and elucidate their roles in CAD. The resulting PPI networks contain interactions that are outside of known biology in the vasculature and are enriched for genes involved in immunity-related and arterial-wall-specific mechanisms. Several PPI networks derived from smooth muscle cells are significantly enriched for genetic variants associated with CAD and related vascular phenotypes. Furthermore, the networks identify 61 genes that are found in genetic loci associated with risk of CAD, prioritizing them as the causal candidates within these loci. These findings indicate that the PPI networks we have generated are a rich resource for guiding future research into the molecular pathogenesis of CAD.

Assessment of the alpha 7 nicotinic acetylcholine receptor as an imaging marker of cardiac repair-associated processes using NS14490

BackgroundCardiac repair and remodeling following myocardial infarction (MI) is a multifactorial process involving pro-reparative inflammation, angiogenesis and fibrosis. Noninvasive imaging using a radiotracer targeting these processes could be used to elucidate cardiac wound healing mechanisms. The alpha7 nicotinic acetylcholine receptor (ɑ7nAChR) stimulates pro-reparative macrophage activity and angiogenesis, making it a potential imaging biomarker in this context. We investigated this by assessing in vitro cellular expression of ɑ7nAChR, and by using a tritiated version of the PET radiotracer [18F]NS14490 in tissue autoradiography studies.Resultsɑ7nAChR expression in human monocyte-derived macrophages and vascular cells showed the highest relative expression was within macrophages, but only endothelial cells exhibited a proliferation and hypoxia-driven increase in expression. Using a mouse model of inflammatory angiogenesis following sponge implantation, specific binding of [3H]NS14490 increased from 3.6 ± 0.2 µCi/g at day 3 post-implantation to 4.9 ± 0.2 µCi/g at day 7 (n = 4, P < 0.01), followed by a reduction at days 14 and 21. This peak matched the onset of vessel formation, macrophage infiltration and sponge fibrovascular encapsulation. In a rat MI model, specific binding of [3H]NS14490 was low in sham and remote MI myocardium. Specific binding within the infarct increased from day 14 post-MI (33.8 ± 14.1 µCi/g, P ≤ 0.01 versus sham), peaking at day 28 (48.9 ± 5.1 µCi/g, P ≤ 0.0001 versus sham). Histological and proteomic profiling of ɑ7nAChR positive tissue revealed strong associations between ɑ7nAChR and extracellular matrix deposition, and rat cardiac fibroblasts expressed ɑ7nAChR protein under normoxic and hypoxic conditions.Conclusionɑ7nAChR is highly expressed in human macrophages and showed proliferation and hypoxia-driven expression in human endothelial cells. While NS14490 imaging displays a pattern that coincides with vessel formation, macrophage infiltration and fibrovascular encapsulation in the sponge model, this is not the case in the MI model where the ɑ7nAChR imaging signal was strongly associated with extracellular matrix deposition which could be explained by ɑ7nAChR expression in fibroblasts. Overall, these findings support the involvement of ɑ7nAChR across several processes central to cardiac repair, with fibrosis most closely associated with ɑ7nAChR following MI.

Bioactive layered double hydroxide nanoparticles loaded calcein under GelMA scaffolds promoted osteogenesis and angiogenesis for bone regeneration

In regenerative medicine and tissue engineering, the repair and replacement of damaged or lost bone tissue remains an enduring challenge. Traditional approaches often fail to achieve desired outcomes, but recent advancements in material science have introduced innovative solutions to this issue. Gelatin methacryloyl (GelMA), which is derived from gelatin, has a unique combination of biocompatibility and tunable physical properties for bone tissue engineering applications. In this study, a novel composite was constructed to combine the characteristics of GelMA and bioactive layered double hydroxide (LDH) loaded calcein. The GelMA-LDH-calcein (GLC) scaffold is designed to promote the osteogenic differentiation of mesenchymal stem cells (MSCs) and provide a microenvironment conducive to osteogenic differentiation, ultimately increasing the osteogenic activity to support bone regeneration. Our data demonstrated that GLC promoted the osteogenic differentiation related gene (ALP, Runx2, and OPG) expression in bone marrow-derived MSCs (BMSCs) and angiogenesis of human vascular epithelial cells (HUVECs), making it particularly valuable in the application of bone regeneration in vivo. Through the design, synthesis, and optimization of GLC composites, the optimized scaffolds can provide structure supporting and biological activity for cell proliferation, which promoted bone defect regeneration and could be used for further clinical applications.

Profiling human brain vascular cells using single-cell transcriptomics and organoids.

Angiogenesis and neurogenesis are functionally interconnected during brain development. However, the study of the vasculature has trailed other brain cell types because they are delicate and of low abundance. Here we describe a protocol extension to purify prenatal human brain endothelial and mural cells with FACS and utilize them in downstream applications, including transcriptomics, culture and organoid transplantation. This approach is simple, efficient and generates high yields from small amounts of tissue. When the experiment is completed within a 24 h postmortem interval, these healthy cells produce high-quality data in single-cell transcriptomics experiments. These vascular cells can be cultured, passaged and expanded for many in vitro assays, including Matrigel vascular tube formation, microfluidic chambers and metabolic measurements. Under these culture conditions, primary vascular cells maintain expression of cell-type markers for at least 3 weeks. Finally, we describe how to use primary vascular cells for transplantation into cortical organoids, which captures key features of neurovascular interactions in prenatal human brain development. In terms of timing, tissue processing and staining requires ~3 h, followed by an additional 3 h of FACS. The transplant procedure of primary, FACS-purified vascular cells into cortical organoids requires an additional 2 h. The time required for different transcriptomic and epigenomic protocols can vary based on the specific application, and we offer strategies to mitigate batch effects and optimize data quality. In sum, this vasculo-centric approach offers an integrated platform to interrogate neurovascular interactions and human brain vascular development.

Proteomics signatures of cerebrovascular pathology in TGFß1 overexpressing mice

AbstractBackgroundVascular cognitive impairment and dementia (VCID), the second most prevalent of the age‐related dementias, develops as a consequence of various types of cerebrovascular insults that damage brain function (Corriveau et al. 2016). Accumulating lines of evidence point to a link between VCID, both sporadic (Kim et al. 2006) and genetic (Hara et al. 2009, Zellner et al. 2018) forms, and the transforming growth factor beta (TGFB) family signaling. Transgenic mice overexpressing a constitutively active form of TGFβ1 in the brain (TGF mice) recap the cerebrovascular pathology seen in VCID (Wyss‐Coray et al. 2000, Tong et al. 2015), and develop VCID when submitted to a comorbid cardiovascular risk factor for dementia (Trigiani et al. 2020). Our aim was to characterize the cerebrovascular proteome of TGF mice using mass spectrometry‐based quantitative proteomics.MethodEighteen, 6‐month‐old‐TGF and ‐wildtype (WT) mice (N = 9/group) were transcardially perfused and pial arteries harvested under a dissecting microscope. Arterial proteins were extracted, trypsin‐digested, fractionated by strong cation exchange (SCX, gel‐free method) and analyzed by nanoLC‐MS/MS using nanoAcquity UPLC and ESI‐LTQ Orbitrap. For total and/or differentially‐expressed proteins (≥2‐fold change, p≤0.01) we i) performed characterization of proteins, and demonstrated presence of ii) protein RNA‐transcript in mouse and human brain vascular cells using transcriptomics datasets, and iii) identified proteins present in human‐extracellular‐vesicles (EVs) using Vesiclepedia.ResultWe identified 3602 proteins in brain vessels of WT and TGF mice, including canonical vascular proteins (e.g. Claudin‐5), and 103 direct (N = 103) and indirect (N = 1,942) interactors of TGFβ1 (Fig1). We also identified 83 proteins demonstrating significantly altered levels in TGF mice. Level dysregulation in these proteins point to perturbations in brain vessel vasomotricity, remodeling, and inflammation. We further demonstrated that several of the differentially‐expressed mouse proteins are i) expressed in the human brain vasculature, and ii) found as cargo proteins in EVs.ConclusionWe characterized the deleterious impact of TGFβ1 overproduction on the cerebrovascular proteome. Given the growing popularity of extracellular vesicles in blood as a novel and minimally invasive biomarker discovery platform for the age‐related dementias, including VCID, several of the proteins identified by us can serve as protein biomarkers in human.

Single cell multi‐omics implicates immune and vascular cells in Alzheimer’s etiology

AbstractBackgroundUnderstanding the genetic etiology of Alzheimer’s disease (AD) is critical to inform effective therapies but remains a challenge. Genetic heritability of late‐onset AD is ∼60‐80%, and genome‐wide association studies (GWAS) have uncovered dozens of noncoding single nucleotide polymorphisms (SNPs) that influence AD risk. Two challenges for the functional interpretation of AD genetic risk are (1) determining the cell types in which SNPs operate and (2) identifying the genes they dysregulate to drive AD pathogenesis. Single‐nucleus sequencing studies of human neurons, microglia, and other brain cell types have begun addressing these challenges. Yet, many AD risk SNPs remain unmapped. One possibility is that they are expressed in immunovascular brain cell types largely missed by current methods.MethodHere, we optimized Vessel Isolation and Nuclei Extraction for Sequencing (VINE‐seq) to capture multimodal gene expression and chromatin accessibility assays across all major brain cell types from 30 cognitively normal, MCI, and AD human prefrontal cortex samples.ResultWe discover early gene dysregulation in immune and vascular cell types along AD progression. We highlight the gene regulatory networks mediating this dysregulation and implicate processes associated with AD pathology and cognitive decline. Finally, we map noncoding AD risk variants to diverse immune and vascular cell types, thereby expanding our understanding of AD etiology.ConclusionDysfunction of diverse human brain immune and vascular cell types mediate the genetic risk for Alzheimer’s disease.

Human placental vascular and perivascular cell heterogeneity differs between first trimester and term, and in pregnancies affected by foetal growth restriction.

Growth-restricted placentae have a reduced vascular network, impairing exchange of nutrients and oxygen. However, little is known about the differentiation events and cell types that underpin normal/abnormal placental vascular formation and function. Here, we used 23-colour flow cytometry to characterize placental vascular/perivascular populations between first trimester and term, and in foetal growth restriction (FGR). First-trimester endothelial cells had an immature phenotype (CD144+/lowCD36-CD146low), while term endothelial cells expressed mature endothelial markers (CD36+CD146+). At term, a distinct population of CD31low endothelial cells co-expressed mesenchymal markers (CD90, CD26), indicating a capacity for endothelial to mesenchymal transition (EndMT). In FGR, compared with normal pregnancies, endothelial cells constituted 3-fold fewer villous core cells (P < 0.05), contributing to an increased perivascular: endothelial cell ratio (2.6-fold, P < 0.05). This suggests that abnormal EndMT may play a role in FGR. First-trimester endothelial cells underwent EndMT in culture, losing endothelial (CD31, CD34, CD144) and gaining mesenchymal (CD90, CD26) marker expression. Together this highlights how differences in villous core cell heterogeneity and phenotype may contribute to FGR pathophysiology across gestation.

Abstract 13022: Myocardial Restoration and Functional Recovery Through Transplantation of Vascularized Cardiac Microtissues Derived From Human Induced Pluripotent Stem Cells in a Rat Model of Myocardial Infarction

Introduction: Cardiac regenerative therapy is acknowledged as a promising strategy for treating individuals suffering from severe heart failure. To address the current constraints of stem cell therapy, which primarily relies on indirect paracrine effects for functional improvement, the ideal goal would be to restore the damaged myocardium in the diseased heart. To achieve this, the transplantation of biomimetic heart tissues that closely resemble the natural structure of the heart is highly anticipated. Methods: We successfully generated 3-dimensional vascularized cardiac microtissues (VCMs) using human induced pluripotent stem cells (hiPSCs) through dynamic culture on thin cardiac tissue sheets (CTSs) composed of hiPSC-derived cardiomyocytes and vascular cells. These VCMs exhibited a robust vascular network comprising human vascular cells. Subsequently, we transplanted CTSs (n=6) or VCMs (n=6) onto an athymic rat model of myocardial infarction (MI), while a sham operation was performed on another group (n=6). To assess cardiac function, we employed cardiac magnetic resonance imaging. The survival of grafts, extension of fibrotic scars, neovascularization in the MI border zone, and the formation of a human vascular network within the engrafted region were evaluated through histopathology and light sheet fluorescence microscopy with tissue clearing techniques. Results: Functional analyses demonstrated that the VCM group exhibited the highest left ventricular ejection fraction (Sham: 36.5±0.9%, CTS: 43.5±2.9%, VCM: 55.2±2.5%, p &lt; 0.01). Histopathological evaluation revealed that the VCM group had a larger engraftment area, along with the smallest scar extension and the highest vascular density in the myocardial infarction border zone. Furthermore, LSFM revealed the presence of vascular networks originating from human cells within the engrafted VCMs. Conclusions: The transplantation of hiPSC-derived VCMs demonstrated remarkable therapeutic potential in a rat model of MI, leading to the formation of a vascular network by human cells. This approach might hold great promise as an effective strategy for efficiently restoring the damaged myocardium in hearts afflicted with disease.

Controlled release of therapeutic antibody using hydrolytically degradable microgels.

Monoclonal antibodies have gained significant interest as potential therapeutics for treating various diseases. However, these therapies are not always effective due to poor treatment compliance associated with multiple administrations and drug resistance. Thus, there is a growing interest in developing advanced monoclonal antibody delivery systems that can customize pharmacokinetics to enhance therapeutic outcomes. This work aimed to engineer hydrolytic 4-arm PEG maleimide (PEG-4MAL) microgels for the controlled delivery of therapeutic antibodies, specifically anti-angiogenic bevacizumab, to overcome the limitations of current monoclonal antibody therapies. Through a PEGylation reaction with a thiol-terminated PEG linker, the antibody was covalently conjugated to the macromer backbone before microgel synthesis. The PEGylation reaction was simple, effective, and did not affect antibody bioactivity. Antibody release kinetics was tuned by changing the concentration of the hydrolytic linker (0-2 mM) and/or PEG-4MAL:protein molar ratio (1000:1, 2000:1, and 5000:1) in the macromer precursor solution during microgel fabrication. The bioactivity of the released antibody was assessed on human umbilical endothelial vascular cells (HUVEC), demonstrating that extracts from hydrolytic microgels reduced cell proliferation over time. Collectively, this study demonstrates the development of highly tunable delivery platform based on degradable PEG-4MAL microgels that can be adapted for therapeutic antibody-controlled release.

Effects of Acetyl-L-Carnitine on Oxidative Stress in Amyotrophic Lateral Sclerosis Patients: Evaluation on Plasma Markers and Members of the Neurovascular Unit.

Oxidative stress, the alteration of mitochondrial function, and the neurovascular unit (NVU), play a role in Amyotrophic Lateral Sclerosis (ALS) pathogenesis. We aimed to demonstrate the changes in the plasma redox system and nitric oxide (NO) in 32 new ALS-diagnosed patients in treatment with Acetyl-L-Carnitine (ALCAR) compared to healthy controls. We also evaluated the effects of plasma on human umbilical cord-derived endothelial vascular cells (HUVEC) and astrocytes. The analyses were performed at the baseline (T0), after three months (T1), and after six months (T2). In ALS patients at T0/T1, the plasma markers of lipid peroxidation, thiobarbituric acid reactive substances (TBARS) and 4-hydroxy nonenal (4-HNE) were higher, whereas the antioxidants, glutathione (GSH) and the glutathione peroxidase (GPx) activity were lower than in healthy controls. At T2, plasma TBARS and 4-HNE decreased, whereas plasma GSH and the GPx activity increased in ALS patients. As regards NO, the plasma levels were firmly lower at T0-T2 than those of healthy controls. Cell viability, and mitochondrial membrane potential in HUVEC/astrocytes treated with the plasma of ALS patients at T0-T2 were reduced, while the oxidant release increased. Those results, which confirmed the fundamental role of oxidative stress, mitochondrial function, and of the NVU in ALS pathogenesis, can have a double meaning, acting as disease markers at baseline and potential markers of drug effects in clinical practice and during clinical trials.