Search

Despite the suppression of human immunodeficiency virus (HIV) replication by combined antiretroviral therapy (cART), 50–60% of HIV-infected patients suffer from HIV-associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in the central nervous system (CNS) due to HIV infection. We investigated links among circulating plasma exosomal (crExo) proteins and neuropathogenesis in simian/human immunodeficiency virus (SHIV)-infected rhesus macaques (RM) and HIV-infected and cART treated patients (Patient-Exo). Isolated EVs from SHIV-infected (SHIV-Exo) and uninfected (CTL-Exo) RM were predominantly exosomes (particle size < 150 nm). Proteomic analysis quantified 5654 proteins, of which 236 proteins (~4%) were significantly, differentially expressed (DE) between SHIV-/CTL-Exo. Interestingly, different CNS cell specific markers were abundantly expressed in crExo. Proteins involved in latent viral reactivation, neuroinflammation, neuropathology-associated interactive as well as signaling molecules were expressed at significantly higher levels in SHIV-Exo than CTL-Exo. However, proteins involved in mitochondrial biogenesis, ATP production, autophagy, endocytosis, exocytosis, and cytoskeleton organization were significantly less expressed in SHIV-Exo than CTL-Exo. Interestingly, proteins involved in oxidative stress, mitochondrial biogenesis, ATP production, and autophagy were significantly downregulated in primary human brain microvascular endothelial cells exposed with HIV+/cART+ Patient-Exo. We showed that Patient-Exo significantly increased blood–brain barrier permeability, possibly due to loss of platelet endothelial cell adhesion molecule-1 protein and actin cytoskeleton structure. Our novel findings suggest that circulating exosomal proteins expressed CNS cell markers—possibly associated with viral reactivation and neuropathogenesis—that may elucidate the etiology of HAND. [ABSTRACT FROM AUTHOR]

R1918 --> 710.1 --> Background: Aging is an unavoidable stress with ever‐increasing detrimental effects on the brain microvasculature, which affects neuronal health/function and adds vulnerability to strokes and neurological diseases. In this study, we performed an extensive examination of proteins involved in the structure and function of mouse brain cortical microvessels (MVs) using a discovery‐based quantitative proteomic approach. Methods: Cortical MVs were isolated from age‐matched, male and female, young (4–6 months), middle‐aged (12–14 months), and old (20–21 months) mice obtained from Jackson Laboratory [Tg(Thy1‐EGFP)MJrs/J] (Jax No. 007788) and bred in a C57B16J background. The presence of end‐arterioles, capillaries, and venules in MVs was confirmed by light microscopy and by alkaline phosphate staining. Proteomics analysis was performed using liquid chromatography/mass spectrometry. Results: Most differentially expressed (DE) proteins (> 90%) showed no significant disparities between the sexes; however, some significant DE proteins showing sexual differences in MVs decreased from percentage in young (8.3%), to middle‐aged (3.7%), to old (0.5%) mice. Therefore, we combined male and female data for age‐dependent comparisons but noted sex differences for examination. Key proteins involved in the oxidative stress response, mRNA or protein stability, basement membrane (BM) composition, aerobic glycolysis, and mitochondrial function were significantly altered with aging. Relative abundance of superoxide dismutase‐1/‐2, catalase, and thioredoxin were reduced with aging. Proteins participating in either mRNA degradation or pre‐mRNA splicing were significantly increased in old mice MVs, whereas protein stabilizing proteins decreased. Glycolytic proteins were not affected in middle age, but the relative abundance decreased in MVs of old mice. Although most of the 41 examined proteins composing mitochondrial Complexes I‐V were reduced in old mice, six of these proteins showed a significant reduction in middle‐aged mice, but the relative abundance increased in fourteen proteins. Nidogen, collagen, and laminin family members as well as perlecan showed differing patterns during aging, indicating BM reorganization starting in middle age. Conclusions: We suggest that increased oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability, BM integrity, and ATP synthesis capacity. [ABSTRACT FROM AUTHOR]

R4196 --> Background: Brain microvessels (MVs) may represent a more vulnerable segment of the cerebral circulation than large arteries during aging, diabetes, and/or minor strokes. Adverse changes in MVs have been implicated in the development of cognitive impairment, vascular dementia, and Alzheimer's disease. However, development of effective therapies has been hampered by lack of understanding of the underlying mechanisms that negatively affect MVs compared to large arteries. The purpose of this study was to identify potential mechanisms underlying the vulnerability of MVs compared to large arteries to aging and pathological conditions and the impact of sex using quantitative RNA sequencing (RNAseq). Methods: Cerebral arteries (anterior and middle cerebral, basilar, circle of Willis) and cortical MVs (end arterioles, capillaries, venules) were isolated from young age‐matched male and female C57BL6J mice. Arteries and MVs were then used for 3 vs. 3 quantitative RNAseq. RNASeq analyses were performed using the Illumina NextSeq 550 sequencing system. RNASeq expression analysis was performed using Kallisto (v0.46.0), and Sleuth (v0.30.0) with the mouse transcriptome index. Results: For females, the RNAseq analysis identified 14,325 genes out of 51,661genes examined with significant (p < 0.05) differences between arteries and cortical MVs. The greatest differences (3 to 6 times) were seen in the expression of the following genes: Thrombospondin‐2 (calcium ion binding, negative regulation of angiogenesis), SPARC‐related modular calcium‐binding gene 1, Megakaryocyte‐associated tyrosine protein‐kinase (ATP binding), Prostaglandin‐H2 D‐isomerase (fatty acid synthesis), Solute carrier family 7 member 2, SOGA family member 3, Gelsolin (aging and apoptotic process), potassium voltage‐gated channel subunits, and glutamate receptor subunits (Gria, Grin). While most of the Gria, Grin and potassium channels genes were abundantly expressed in MVs, thrombospondins, prostaglandins and apoptosis‐related genes were more highly expressed in large arteries. The RNAseq profiles comparing arteries to MVs for males were very different than for females (extracellular matrix binding, regulation of smooth muscle contraction and growth factors, mostly). Conclusion: We found substantial differences in the gene expression of brain large arteries and cortical MVs in mice and these differences may account for increased vulnerability of smaller blood vessels to aging and stress. For example, increased expression of glutamatergic receptors in MVs might be associated with enhanced vulnerability to damage and dysfunction of this segment of the cerebral circulation due to stress. The finding that RNAseq profiles comparing large arteries and cortical MVs differ between males and females may provide the basis for sex‐dependent differences in the vulnerability and severity of neurological disorders in humans. [ABSTRACT FROM AUTHOR]

R4023 --> Background: The relationship between gene expression and corresponding protein levels are complex due to many factors. Therefore, we compared multiomics (RNA‐Seq and proteomic) results to discover similarities and differences. Although RNA‐Seq and proteomic approaches have been applied independently to the field of microvascular pathophysiology, to date there are no reported studies of sex‐dependent differences in rat cerebral microvessels (MVs) by multiomics analyses. Methods: Cerebral MVs were isolated from 8‒10 weeks old, male and female, Sprague‐Dawley rats, and the presence of end‐arterioles, capillaries, and venules in MVs was confirmed by light microscopy and by alkaline phosphate staining. RNA‐Seq analysis was performed by Illumina NextSeq 550 sequencing system. Proteomics analysis was performed, using liquid chromatography/mass spectrometry (LC‐MS/MS). The expression of top genes and proteins were validated by RT‐qPCR and western blotting, respectively. Results: In RNA‐Seq analysis, among the 23,786 identified genes, a total of 298 (1.2%) significant (FDR < 0.05) differentially expressed (DE) genes were identified, of which 40% and 60% of DE genes were abundant in male and female MVs, respectively. Instead, among the 1,871 quantified proteins, a total of 198 (10.6%) significant DE proteins were quantified, of which 84% and 16% of DE proteins were abundant in male and female MVs, respectively. Nucleic acid binding, enzyme modulator, and transcription factor were the top three gene functions in male MVs. Whereas cytoskeletal protein, extracellular matrix protein, and transcriptional regulator were the top three gene functions in female MVs. Gene functions were supported by proteomic analysis. The gene‐centric top three canonical pathways in male MVs involved glycosylphosphatidylinositol (GPI), biosynthesis, and post‐translational modification (PTM) of GPI‐anchored proteins. Similarly, mitochondrial respiration, ribosome, and 3´UTR mediated translational regulation pathways were the top gene‐centric canonical pathways in female MVs. Surprisingly, despite large differences in the expression of some genes, the resultant proteins are not always consistent with these differences. There was a greater correlation between gene expression and protein synthesis in female MVs while, despite relatively large differences in gene expression favoring males, actual protein levels were either not different between the sexes or were opposite of expectations. Genes related with PTM and ubiquitin‐mediated proteolysis were highly expressed in male MVs and probably play an important role on the formation, stability, and functions of various proteins. Conclusions: We have provided the first comprehensive multiomics analysis of male and female MVs of rats. Our study indicates that male and female MVs have significant differences in gene expression and protein synthesis, which may explain the sex‐dependent differences in the microcirculation during health and diseases. [ABSTRACT FROM AUTHOR]