Aging Alters the Aortic Proteome in Health and Thoracic Aortic Aneurysm.

BACKGROUND: Aging enhances most chronic diseases but its impact on human aortic tissue in health and in thoracic aortic aneurysms (TAA) remains unclear. METHODS: We employed a human aortic biorepository of healthy specimens (n=17) and those that underwent surgical repair for TAA (n=20). First, we performed proteomics comparing aortas of healthy donors to aneurysmal specimens, in young (ie, <60 years of age) and old (ie, ≥60 years of age) subjects. Second, we measured proteins, via immunoblotting, involved in mitophagy (ie, Parkin) and also mitochondrial-induced inflammatory pathways, specifically TLR (toll-like receptor) 9, STING (stimulator of interferon genes), and IFN (interferon)-ß. RESULTS: Proteomics revealed that aging transformed the aorta both quantitatively and qualitatively from health to TAA. Whereas young aortas exhibited an enrichment of immunologic processes, older aortas exhibited an enrichment of metabolic processes. Immunoblotting revealed that the expression of Parkin directly correlated to subject age in health but inversely to subject age in TAA. In TAA, but not in health, phosphorylation of STING and the expression of IFN-ß was impacted by aging regardless of whether subjects had bicuspid or tricuspid valves. In subjects with bicuspid valves and TAAs, TLR9 expression positively correlated with subject age. Interestingly, whereas phosphorylation of STING was inversely correlated with subject age, IFN-ß positively correlated with subject age. CONCLUSIONS: Aging transforms the human aortic proteome from health to TAA, leading to a differential regulation of biological processes. Our results suggest that the development of therapies to mitigate vascular diseases including TAA may need to be modified depending on subject age.

Long-term multimodal imaging characterization of persistent retinal neovascularization using DL-alpha-aminoadipic acid in pigmented and white rabbits.

PURPOSE: Intravitreal (IVT) injection of DL-alpha-aminoadipic acid (AAA) is a new animal model for retinal neovascularization (RNV) reported in rabbits. This study performs longitudinal multimodal imaging for up to 1 year to evaluate DL-AAA RNV in both New Zealand white (NZW) rabbits and Dutch-Belted pigmented (DBP) rabbits. METHOD: Detailed characterization and quantification of this model were performed in these two strains in 32 eyes by optical coherence tomography (OCT), fundus photography, and fluorescein angiography (FA) for up to 16 weeks following DL-AAA administration in 32 eyes and up to 52 weeks in 5 eyes. H & E histology was also performed in these two strains 8 weeks after injection of DL-AAA. RESULT: RNV was successfully generated using 50 µL 80 mM DL-AAA solution for DBP rabbits and 80 µL 80 mM DL-AAA for NZW rabbits. The incidence of persistent vascular leakage is 100% (15/15) for DBP rabbits and 70.6% (12/17) for NZW rabbits at 16 weeks. Complications with NZW rabbits ultimately decreased the efficiency in NZW rabbits to 58.8% (10/17) of NZW rabbits getting persistent (to 16 weeks) vascular leakage without ocular complications as compared with 100% (15/15) in DBP rabbits. Five eyes (2 DBP and 3 NZW) were selected from those demonstrating RNV at 16 weeks and were monitored for up to 52 weeks. All 5 demonstrated persistent RNV to 52 weeks. Quantification of the mean leakage area (MLA) in DBP rabbits is more accurate than in NZW rabbits since the reduced contrast between the leakage and background in NZW rabbits makes it more challenging to quantify. CONCLUSION: DL-AAA can induce persistent and quantifiable RNV in both DBP and NZW rabbits. DBP rabbits have a higher success rate, lower required volume of DL-AAA, and more accurate method for quantification that could be more desirable.

In Vitro Lineage-Specific Differentiation of Vascular Smooth Muscle Cells in Response to SMAD3 Deficiency: Implications for SMAD3-Related Thoracic Aortic Aneurysm.

OBJECTIVE: SMAD3 pathogenic variants are associated with the development of thoracic aortic aneurysms. We sought to determine the role of SMAD3 in lineage-specific vascular smooth muscle cells (VSMCs) differentiation and function. Approach and Results: SMAD3 c.652delA, a frameshift mutation and nonsense-mediated decay, was introduced in human-induced pluripotent stem cells using CRISPR-Cas9. The wild-type and SMAD3-/- (c.652delA) human-induced pluripotent stem cells were differentiated into cardiovascular progenitor cells or neural crest stem cells and then to lineage-specific VSMCs. Differentiation, contractility, extracellular matrix synthesis, and TGF-ß (transforming growth factor-ß) signaling of the differentiated VSMCs were analyzed. The homozygous frameshift mutation resulted in SMAD3 deficiency and was confirmed in human-induced pluripotent stem cells by Sanger sequencing and immunoblot analysis. In cardiovascular progenitor cell-VSMCs, SMAD3 deletion significantly disrupted canonical TGF-ß signaling and decreased gene expression of VSMC markers, including SM α-actin, myosin heavy chain 11, calponin-1, SM22α, and key controlling factors, SRF and myocardin, but increased collagen expression. The loss of SMAD3 significantly decreased VSMC contractility. In neural crest stem cells-VSMCs, SMAD3 deficiency did not significantly affect the VSMC differentiation but decreased ELN (elastin) expression and increased phosphorylated SMAD2. Expression of mir-29 was increased in SMAD3-/- VSMCs, and inhibition of mir-29 partially rescued ELN expression. CONCLUSIONS: SMAD3-dependent TGF-ß signaling was essential for the differentiation of cardiovascular progenitor cell-VSMCs but not for the differentiation of neural crest stem cell-VSMCs. The lineage-specific TGF-ß responses in human VSMCs may potentially contribute to the development of aortic root aneurysms in patients with SMAD3 mutations.

Identification of immune-related hub genes and analysis of infiltrated immune cells of idiopathic pulmonary artery hypertension.

Objectives: Idiopathic pulmonary artery hypertension (IPAH) is a rare but life-threaten disease. However, the mechanism underlying IPAH is unclear. In this study, underlying mechanism, infiltration of immune cells, and immune-related hub genes of IPAH were analyzed via bioinformatics. Methods: GSE15197, GSE48149, GSE113439, and GSE117261 were merged as lung dataset. Weighted gene correlation network analysis (WGCNA) was used to construct the co-expression gene networks of IPAH. Gene Ontology and pathway enrichment analysis were performed using DAVID, gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA). Infiltration of immune cells in lung samples was analyzed using CIBERSORT. GSE22356 and GSE33463 were merged as peripheral blood mononuclear cells (PBMCs) dataset. Immune-related differentially expressed genes (IRDEGs) of lung and PBMCs dataset were analyzed. Based on the intersection between two sets of IRDEGs, hub genes were screened using machine learning algorithms and validated by RT-qPCR. Finally, competing endogenous RNA (ceRNA) networks of hub genes were constructed. Results: The gray module was the most relevant module and genes in the module enriched in terms like inflammatory and immune responses. The results of GSEA and GSVA indicated that increasement in cytosolic calcium ion, and metabolism dysregulation play important roles in IPAH. The proportions of T cells CD4 memory resting and macrophage M1 were significantly greater in IPAH group, while the proportions of monocytes and neutrophils were significantly lower in IPAH group. IRDEGs of two datasets were analyzed and the intersection between two set of IRDEGs were identified as candidate hub genes. Predictive models for IPAH were constructed using data from PBMCs dataset with candidate hub genes as potential features via LASSO regression and XGBoost algorithm, respectively. CXCL10 and VIPR1 were identified as hub genes and ceRNA networks of CXCL10 was constructed. Conclusion: Inflammatory response, increasement in cytosolic calcium ion, and metabolism dysregulation play important roles in IPAH. T cells CD4 memory resting and macrophage M1 were significantly infiltrated in lung samples from patients with IPAH. IRDEGs of lung dataset and PBMCs dataset were analyzed, and CXCL10 and VIPR1 were identified as hub genes.

Identification of biomarkers and analysis of infiltrated immune cells in stable and ruptured abdominal aortic aneurysms.

Objectives: The mortality rate of abdominal aortic aneurysm (AAA) is extremely high in the older population. This study aimed to identify potential biomarkers of AAA and aortic rupture and analyze infiltration of immune cells in stable and ruptured AAA samples. Methods: Raw data of GSE47472, GSE57691, and GSE98278 were downloaded. After data processing, the co-expression gene networks were constructed. Gene Ontology and pathway enrichment analysis of AAA- and aortic rupture-related gene modules were conducted using the Database for Annotation, Visualization, and Integrated Discovery. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used for further enrichment analysis. The CIBERSORT tool was used to analyze the relative abundance of immune cells in samples. Differentially expressed immune-related genes were analyzed between different samples. Predictive models were constructed via extreme gradient boosting, and hub genes were identified according to feature importance. Results: Blue and yellow modules were significantly related to AAA, and genes in these modules were associated with the aortic wall and immune response, respectively. In terms of aortic rupture, the most relevant module was significantly enriched in the inflammatory response. The results of GSEA and GSVA suggested that immune cells and the inflammatory response were involved in the development of AAA and aortic rupture. There were significant differences in the infiltration of immune cells and expression levels of immune-related genes among different samples. NFKB1 might be an important transcription factor mediating the inflammatory response of AAA and aortic rupture. After the construction of a predictive model, CD19, SELL, and CCR7 were selected as hub genes for AAA whereas OAS3, IFIT1, and IFI44L were identified as hub genes for aortic rupture. Conclusion: Weakening of the aortic wall and the immune response both contributed to the development of AAA, and the inflammatory response was closely associated with aortic rupture. The infiltration of immune cells was significantly different between different samples. NFKB1 might be an important transcription factor in AAA and aortic rupture. CD19, SELL, and CCR7 had potential diagnostic value for AAA. OAS3, IFIT1, and IFI44L might be predictive factors for aortic rupture.

Biomimetic tubular scaffold with heparin conjugation for rapid degradation in in situ regeneration of a small diameter neoartery.

To address the clinical need for readily available small diameter vascular grafts, biomimetic tubular scaffolds were developed for rapid in situ blood vessel regeneration. The tubular scaffolds were designed to have an inner layer that is porous, interconnected, and with a nanofibrous architecture, which provided an excellent microenvironment for host cell invasion and proliferation. Through the synthesis of poly(spirolactic-co-lactic acid) (PSLA), a highly functional polymer with a norbornene substituting a methyl group in poly(l-lactic acid) (PLLA), we were able to covalently attach biomolecules onto the polymer backbone via thiol-ene click chemistry to impart desirable functionalities to the tubular scaffolds. Specifically, heparin was conjugated on the scaffolds in order to prevent thrombosis when implanted in situ. By controlling the amount of covalently attached heparin we were able to modulate the physical properties of the tubular scaffold, resulting in tunable wettability and degradation rate while retaining the porous and nanofibrous morphology. The scaffolds were successfully tested as rat abdominal aortic replacements. Patency and viability were confirmed through dynamic ultrasound and histological analysis of the regenerated tissue. The harvested tissue showed excellent vascular cellular infiltration, proliferation, and migration with laminar cellular arrangement. Furthermore, we achieved both complete reendothelialization of the vessel lumen and native-like media extracellular matrix. No signs of aneurysm or hyperplasia were observed after 3 months of vessel replacement. Taken together, we have developed an effective vascular graft able to generate small diameter blood vessels that can function in a rat model.