Cardiovascular-related proteomic changes in ECFCs exposed to the serum of COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection significantly affects the cardiovascular system, causing vascular damage and thromboembolic events in critical patients. Endothelial dysfunction represents one of the first steps in response to COVID-19 that might lead to cardiovascular complications and long-term sequelae. However, despite the enormous efforts in the last two years, the molecular mechanisms involved in such processes remain poorly understood. Herein, we analyzed the protein changes taking place in endothelial colony forming cells (ECFCs) after the incubation with the serum from individuals infected with COVID-19, whether asymptomatic or critical patients, by application of a label free-quantitative proteomics approach. Specifically, ECFCs from healthy individuals were incubated ex-vivo with the serum of either COVID-19 negative donors (PCR-/IgG-, n:8), COVID-19 asymptomatic donors at different infective stages (PCR+/ IgG-, n:8and PCR-/IgG+, n:8), or hospitalized critical COVID-19 patients (n:8), followed by proteomics analysis. In total, 590 proteins were differentially expressed in ECFCs in response to all infected serums. Predictive analysis highlighted several proteins like CAPN5, SURF4, LAMP2 or MT-ND1, as highly discriminating features between the groups compared. Protein changes correlated with viral infection, RNA metabolism or autophagy, among others. Remarkably, the angiogenic potential of ECFCs in response to the infected serums was impaired, and many of the protein alterations in response to the serum of critical patients were associated with cardiovascular-related pathologies.

Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach.

BACKGROUND: Endothelial colony forming cells (ECFCs), alone or in combination with mesenchymal stem cells, have been selected as potential therapeutic candidates for critical limb-threatening ischemia (CLTI), mainly for those patients considered as "no-option," due to their capability to enhance revascularization and perfusion recovery of ischemic tissues. Nevertheless, prior to translating cell therapy to the clinic, biodistribution assays are required by regulatory guidelines to ensure biosafety as well as to discard undesired systemic translocations. Different approaches, from imaging technologies to qPCR-based methods, are currently applied. METHODS: In the current study, we have optimized a cell-tracking assay based on DiR fluorescent cell labeling and near-infrared detection for in vivo and ex vivo assays. Briefly, an improved protocol for DiR staining was set up, by incubation of ECFCs with 6.67 µM DiR and intensive washing steps prior cell administration. The minimal signal detected for the residual DiR, remaining after these washes, was considered as a baseline signal to estimate cell amounts correlated to the DiR intensity values registered in vivo. Besides, several assays were also performed to determine any potential effect of DiR over ECFCs functionality. Furthermore, the optimized protocol was applied in combination with qPCR amplification of specific human Alu sequences to assess the final distribution of ECFCs after intramuscular or intravenous administration to a murine model of CLTI. RESULTS: The optimized DiR labeling protocol indicated that ECFCs administered intramuscularly remained mainly within the hind limb muscle while cells injected intravenously were found in the spleen, liver and lungs. CONCLUSION: Overall, the combination of DiR labeling and qPCR analysis in biodistribution assays constitutes a highly sensitive approach to systemically track cells in vivo. Thereby, human ECFCs administered intramuscularly to CLTI mice remained locally within the ischemic tissues, while intravenously injected cells were found in several organs. Our data corroborate the need to perform biodistribution assays in order to define specific parameters such as the optimal delivery route for ECFCs before their application into the clinic.

Serum microRNAs targeting ACE2 and RAB14 genes distinguish asymptomatic from critical COVID-19 patients.

Despite the extraordinary advances achieved to beat COVID-19 disease, many questions remain unsolved, including the mechanisms of action of SARS-CoV-2 and which factors determine why individuals respond so differently to the viral infection. Herein, we performed an in silico analysis to identify host microRNA targeting ACE2, TMPRSS2, and/or RAB14, all genes known to participate in viral entry and replication. Next, the levels of six microRNA candidates previously linked to viral and respiratory-related pathologies were measured in the serum of COVID-19-negative controls (n = 16), IgG-positive COVID-19 asymptomatic individuals (n = 16), and critical COVID-19 patients (n = 17). Four of the peripheral microRNAs analyzed (hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and hsa-miR-1246) were upregulated in COVID-19 critical patients compared with COVID-19-negative controls. Moreover, hsa-miR-32-5p and hsa-miR-1246 levels were also altered in critical versus asymptomatic individuals. Furthermore, these microRNA target genes were related to viral infection, inflammatory response, and coagulation-related processes. In conclusion, SARS-CoV-2 promotes the alteration of microRNAs targeting the expression of key proteins for viral entry and replication, and these changes are associated with disease severity. The microRNAs identified could be taken as potential biomarkers of COVID-19 progression as well as candidates for future therapeutic approaches against this disease.

The serum of COVID-19 asymptomatic patients up-regulates proteins related to endothelial dysfunction and viral response in circulating angiogenic cells ex-vivo.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already caused 6 million deaths worldwide. While asymptomatic individuals are responsible of many potential transmissions, the difficulty to identify and isolate them at the high peak of infection constitutes still a real challenge. Moreover, SARS-CoV-2 provokes severe vascular damage and thromboembolic events in critical COVID-19 patients, deriving in many related deaths and long-hauler symptoms. Understanding how these processes are triggered as well as the potential long-term sequelae, even in asymptomatic individuals, becomes essential. METHODS: We have evaluated, by application of a proteomics-based quantitative approach, the effect of serum from COVID-19 asymptomatic individuals over circulating angiogenic cells (CACs). Healthy CACs were incubated ex-vivo with the serum of either COVID-19 negative (PCR -/IgG -, n:8) or COVID-19 positive asymptomatic donors, at different infective stages: PCR +/IgG - (n:8) and PCR -/IgG + (n:8). Also, a label free quantitative approach was applied to identify and quantify protein differences between these serums. Finally, machine learning algorithms were applied to validate the differential protein patterns in CACs. RESULTS: Our results confirmed that SARS-CoV-2 promotes changes at the protein level in the serum of infected asymptomatic individuals, mainly correlated with altered coagulation and inflammatory processes (Fibrinogen, Von Willebrand Factor, Thrombospondin-1). At the cellular level, proteins like ICAM-1, TLR2 or Ezrin/Radixin were only up-regulated in CACs treated with the serum of asymptomatic patients at the highest peak of infection (PCR + /IgG -), but not with the serum of PCR -/IgG + individuals. Several proteins stood out as significantly discriminating markers in CACs in response to PCR or IgG + serums. Many of these proteins particiArticle title: Kindly check and confirm the edit made in the article title.pate in the initial endothelial response against the virus. CONCLUSIONS: The ex vivo incubation of CACs with the serum of asymptomatic COVID-19 donors at different stages of infection promoted protein changes representative of the endothelial dysfunction and inflammatory response after viral infection, together with activation of the coagulation process. The current approach constitutes an optimal model to study the response of vascular cells to SARS-CoV-2 infection, and an alternative platform to test potential inhibitors targeting either the virus entry pathway or the immune responses following SARS-CoV-2 infection.

Long Term Response to Circulating Angiogenic Cells, Unstimulated or Atherosclerotic Pre-Conditioned, in Critical Limb Ischemic Mice.

Critical limb ischemia (CLI), the most severe form of peripheral artery disease, results from the blockade of peripheral vessels, usually correlated to atherosclerosis. Currently, endovascular and surgical revascularization strategies cannot be applied to all patients due to related comorbidities, and even so, most patients require re-intervention or amputation within a year. Circulating angiogenic cells (CACs) constitute a good alternative as CLI cell therapy due to their vascular regenerative potential, although the mechanisms of action of these cells, as well as their response to pathological conditions, remain unclear. Previously, we have shown that CACs enhance angiogenesis/arteriogenesis from the first days of administration in CLI mice. Also, the incubation ex vivo of these cells with factors secreted by atherosclerotic plaques promotes their activation and mobilization. Herein, we have evaluated the long-term effect of CACs administration in CLI mice, whether pre-stimulated or not with atherosclerotic factors. Remarkably, mice receiving CACs and moreover, pre-stimulated CACs, presented the highest blood flow recovery, lower progression of ischemic symptoms, and decrease of immune cells recruitment. In addition, many proteins potentially involved, like CD44 or matrix metalloproteinase 9 (MMP9), up-regulated in response to ischemia and decreased after CACs administration, were identified by a quantitative proteomics approach. Overall, our data suggest that pre-stimulation of CACs with atherosclerotic factors might potentiate the regenerative properties of these cells in vivo.

REX-001, a BM-MNC Enriched Solution, Induces Revascularization of Ischemic Tissues in a Murine Model of Chronic Limb-Threatening Ischemia.

Background: Bone Marrow Mononuclear Cells (BM-MNC) constitute a promising alternative for the treatment of Chronic Limb-Threatening ischemia (CLTI), a disease characterized by extensive blockade of peripheral arteries, clinically presenting as excruciating pain at rest and ischemic ulcers which may lead to gangrene and amputation. BM-MNC implantation has shown to be efficient in promoting angiogenesis and ameliorating ischemic symptoms in CLTI patients. However, the variability seen between clinical trials makes necessary a further understanding of the mechanisms of action of BM-MNC, and moreover, to improve trial characteristics such as endpoints, inclusion/exclusion criteria or drug product compositions, in order to implement their use as stem-cell therapy. Materials: Herein, the effect of REX-001, a human-BM derived cell suspension enriched for mononuclear cells, granulocytes and CD34+ cells, has been assessed in a murine model of CLTI. In addition, a REX-001 placebo solution containing BM-derived red blood cells (BM-RBCs) was also tested. Thus, 24 h after double ligation of the femoral artery, REX-001 and placebo were administrated intramuscularly to Balb-c nude mice (n:51) and follow-up of ischemic symptoms (blood flow perfusion, motility, ulceration and necrosis) was carried out for 21 days. The number of vessels and vascular diameter sizes were measured within the ischemic tissues to evaluate neovascularization and arteriogenesis. Finally, several cell-tracking assays were performed to evaluate potential biodistribution of these cells. Results: REX-001 induced a significant recovery of blood flow by increasing vascular density within the ischemic limbs, with no cell translocation to other organs. Moreover, cell tracking assays confirmed a decrease in the number of infused cells after 2 weeks post-injection despite on-going revascularization, suggesting a paracrine mechanism of action. Conclusion: Overall, our data supported the role of REX-001 product to improve revascularization and ischemic reperfusion in CLTI.

Atherosclerotic Pre-Conditioning Affects the Paracrine Role of Circulating Angiogenic Cells Ex-Vivo.

In atherosclerosis, circulating angiogenic cells (CAC), also known as early endothelial progenitor cells (eEPC), are thought to participate mainly in a paracrine fashion by promoting the recruitment of other cell populations such as late EPC, or endothelial colony-forming cells (ECFC), to the injured areas. There, ECFC replace the damaged endothelium, promoting neovascularization. However, despite their regenerative role, the number and function of EPC are severely affected under pathological conditions, being essential to further understand how these cells react to such environments in order to implement their use in regenerative cell therapies. Herein, we evaluated the effect of direct incubation ex vivo of healthy CAC with the secretome of atherosclerotic arteries. By using a quantitative proteomics approach, 194 altered proteins were identified in the secretome of pre-conditioned CAC, many of them related to inhibition of angiogenesis (e.g., endostatin, thrombospondin-1, fibulins) and cell migration. Functional assays corroborated that healthy CAC released factors enhanced ECFC angiogenesis, but, after atherosclerotic pre-conditioning, the secretome of pre-stimulated CAC negatively affected ECFC migration, as well as their ability to form tubules on a basement membrane matrix assay. Overall, we have shown here, for the first time, the effect of atherosclerotic factors over the paracrine role of CAC ex vivo. The increased release of angiogenic inhibitors by CAC in response to atherosclerotic factors induced an angiogenic switch, by blocking ECFC ability to form tubules in response to pre-conditioned CAC. Thus, we confirmed here that the angiogenic role of CAC is highly affected by the atherosclerotic environment.

Identification of the initial molecular changes in response to circulating angiogenic cells-mediated therapy in critical limb ischemia.

BACKGROUND: Critical limb ischemia (CLI) constitutes the most aggressive form of peripheral arterial occlusive disease, characterized by the blockade of arteries supplying blood to the lower extremities, significantly diminishing oxygen and nutrient supply. CLI patients usually undergo amputation of fingers, feet, or extremities, with a high risk of mortality due to associated comorbidities. Circulating angiogenic cells (CACs), also known as early endothelial progenitor cells, constitute promising candidates for cell therapy in CLI due to their assigned vascular regenerative properties. Preclinical and clinical assays with CACs have shown promising results. A better understanding of how these cells participate in vascular regeneration would significantly help to potentiate their role in revascularization. Herein, we analyzed the initial molecular mechanisms triggered by human CACs after being administered to a murine model of CLI, in order to understand how these cells promote angiogenesis within the ischemic tissues. METHODS: Balb-c nude mice (n:24) were distributed in four different groups: healthy controls (C, n:4), shams (SH, n:4), and ischemic mice (after femoral ligation) that received either 50 µl physiological serum (SC, n:8) or 5 × 105 human CACs (SE, n:8). Ischemic mice were sacrificed on days 2 and 4 (n:4/group/day), and immunohistochemistry assays and qPCR amplification of Alu-human-specific sequences were carried out for cell detection and vascular density measurements. Additionally, a label-free MS-based quantitative approach was performed to identify protein changes related. RESULTS: Administration of CACs induced in the ischemic tissues an increase in the number of blood vessels as well as the diameter size compared to ischemic, non-treated mice, although the number of CACs decreased within time. The initial protein changes taking place in response to ischemia and more importantly, right after administration of CACs to CLI mice, are shown. CONCLUSIONS: Our results indicate that CACs migrate to the injured area; moreover, they trigger protein changes correlated with cell migration, cell death, angiogenesis, and arteriogenesis in the host. These changes indicate that CACs promote from the beginning an increase in the number of vessels as well as the development of an appropriate vascular network.