Objective: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. Results: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3–4 h. Percoll purification from 100 to 200 mg fresh tissue yielded ~ 200–400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9 to 7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

Pro-angiogenic paracrine/autocrine signaling impacts myocardial repair in cell-based therapies. Activin A receptor-like type 1 (ACVRL1, ALK1) signaling plays a pivotal role in cardiovascular development and maintenance, but its importance in human-derived therapeutic cardiac cells is not well understood. Here, we isolated a subpopulation of human highly proliferative cells (hHiPCs) from adult epicardial tissue and found that they express ALK1, a high affinity receptor for bone morphogenetic protein-9 (BMP9), which signals via SMAD1/5 to regulate paracrine/autocrine signaling and angiogenesis. We show that in humans, circulating BMP9 level is negatively associated with the number of epicardial hHiPC and positively associated with endothelial cell (EC) number in the adult heart, implicating the potential importance of this signaling pathway in cardiac cell fate and vascular maintenance. To investigate BMP9/ALK1 signaling in hHiPCs, we selected a primary cell population of hHiPC from each of 3 individuals and studied their responses to BMP9 and BMP10 treatment in vitro. Proteins were collected in conditioned media (CM) for mass spectrometry and cell-based assays on human ECs and hHiPCs. Proteomic analysis of the hHiPC secretome following BMP9 or BMP10 treatment demonstrates that the secreted proteins, sclerostin (SOST), meflin/immunoglobulin superfamily containing leucine rich repeat (ISLR), and insulin-like growth factor binding protein-3 (IGFBP3), are novel regulated targets of BMP9/ALK1 signaling. Lentiviral shRNA and pharmacological inhibition of ALK1 in hHiPCs suppressed transcription and secretion of SOST, ISLR, and IGFBP3 following BMP9 treatment. Moreover, the BMP9-treated secretome of hHiPC increased capillary-like tube formation of ECs and hHiPCs. Treatment of hHiPCs with recombinant SOST increased VEGF-a expression, increased tube formation and enhanced expression of EC receptor marker annexin A2 (ANXA2). These data provide the first proteomic characterization of hHiPC, identifying BMP9/ALK1-mediated target protein secretion in hHiPCs, and underscore the complex role of BMP9/ALK1 signaling in paracrine/autocrine mediated angiogenesis. Data are available via ProteomeXchange with identifier PXD055302.

Background/Objectives: Perivascular adipose tissue (PVAT) exerts a paracrine effect on blood vessels and our objective was to understand PVAT molecular signatures related to cardiovascular disease. Methods: We studied two groups: those undergoing mitral valve repair/replacement (VR, n = 16) and coronary artery bypass graft (CABG, n = 38). VR donors did not have coronary artery disease, whereas CABG donors had advanced coronary artery disease. Clinical and tissue pathologies and proteomics from adipose tissue were assessed. Results: Donors undergoing VR had a lower body mass index (p = 0.01), HbA1C (p = 0.0023), and incidence of diabetes (p = 0.022) compared to CABG. VR donors were overall healthier, with higher cardiac function compared to CABG donors, based on ejection fraction. Although adipose histopathology between groups was not markedly different, PVAT had smaller and more adipocytes compared to subcutaneous adipose tissues. These differences were validated by whole specimen automated morphological analysis, and anisotropy analysis showed small (2.8–7.5 μm) and large (22.8–64.4 μm) scale differences between perivascular and subcutaneous adipose tissue from CABG donors, and small scale changes (2.8–7.5 μm) between perivascular and subcutaneous adipose tissue from VR donors. Distinct protein signatures in PVAT and subcutaneous adipose tissue include those involved in secretion, exosomes and vesicles, insulin resistance, and adipocyte identity. Comparing PVAT and subcutaneous adipose tissue from CABG donors, there were 82 significantly different proteins identified with log fold change ≥ 0.3 or ≤−0.3 (p < 0.05). Using this threshold, there were 36 differences when comparing PVAT and subcutaneous adipose tissue from VR donors, 58 differences when comparing PVAT from CABG or VR donors, and 55 when comparing subcutaneous adipose tissue from CABG vs. VR donors. Conclusions: Routine histopathology cannot differentiate between PVAT from donors with or without coronary artery disease, but multiscale anisotropy analysis discriminated between these populations. Our mass spectrometry analysis identified a cohort of proteins that distinguish between adipose depots, and are also associated with the presence or absence of coronary artery disease.

Extracellular particles (EPs), an umbrella term encompassing membrane-enclosed extracellular vesicles (EVs) and non-vesicular extracellular particles ([NVEPs], previously described as extracellular condensates [ECs]) contain a complex cargo of biomolecules, including DNA, RNA, proteins, and lipids, reflecting the physiological state of their cell of origin. Identifying proteins associated with EPs that regulate host responses to physiological and pathophysiological processes is of critical importance. Here, we report the findings of our study to gain insight into the proteins associated with NVEPs. We used samples from human semen, the rat brain, and the rhesus macaque (RM) brain and blood to assess the physical properties and proteome profiles of NVEPs from these specimens. The results show significant differences in the zeta potential, concentration, and size of NVEPs across different species. We identified 938, 51, and 509 total proteins from NVEPs isolated from rat brain tissues, RM blood, and human seminal plasma, respectively. The species-specific protein networks show distinct biological themes, while the species-conserved protein interactome was identified with six proteins (ALB, CST3, FIBA/FGA, GSTP1, PLMN/PLG, PPIA) associated with NVEPs in all samples. The six NVEP-associated proteins are prone to aggregation and formation of wide, insoluble, unbranched filaments with a cross-beta sheet quaternary structure, such as amyloid fibrils. Protein-to-function analysis indicates that the six identified proteins are linked to the release of dopamine, immune-mediated inflammatory disease, replication of RNA viruses, HIV-HCV co-infection, and inflammation. These interesting findings have created an opportunity to evaluate NVEPs for their potential use as biomarkers of health and disease. Additional in-depth studies are needed to clarify when and how these proteins sustain their physiological role or transition to pathogenic roles.

Objective: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. Results: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 hours. Percoll purification from 100-200 mg fresh tissue yielded ∼200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9-7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

Adipogenesis is regulated by the coordinated activity of adipogenic transcription factors including PPAR-gamma and C/EBP alpha, while dysregulated adipogenesis can predispose adipose tissues to adipocyte hypertrophy and hyperplasia. We have previously reported that Cthrc1-null mice have increased adiposity compared to wildtype mice, supporting the notion that CTHRC1 regulates body composition. Herein, we derived conditioned medium from 3T3-L1 cells expressing human CTHRC1 and investigated its anti-adipogenic activity. This constituent significantly reduced 3T3-L1 cell adipogenic differentiation commensurate to the marked suppression of Cebpa and Pparg gene expression. It also increased the expression of the anti-adipogenic transcription factor SOX9 and promoted its nuclear translocation. Importantly, Sox9 gene knockdown demonstrated that the anti-adipogenic effect produced by this conditioned medium is dependent on SOX9 expression, while its ability to positively regulate SOX9 was attenuated by the application of Rho and Rac1 signaling pathway inhibitors. We also identified the selective expression of CTHRC1 in PDGFRA-expressing cell populations in human white adipose tissue, but not brown or perivascular adipose tissues. Congruently, flow cytometry revealed CTHRC1 expression in PDGFR-alpha⁺ stromal cells of mouse white adipose tissue, thus defining a novel stromal cell population that could underpin the ability of CTHRC1 to regulate adiposity.