Reverse cholesterol transport (RCT) is considered as the most important antiatherogenic role of high-density lipoproteins (HDL), but interventions based on RCT have failed to reduce the risk of coronary heart disease. In contrast to RCT, important evidence suggests that HDL deliver lipids to peripheral cells. Therefore, in this paper, we investigated whether HDL could improve endothelial function by delivering lipids to the cells. Internalization kinetics using cholesterol and apolipoprotein (apo) AI fluorescent double-labeled reconstituted HDL (rHDL), and human dermal microvascular endothelial cells-1 (HMEC-1) showed a fast cholesterol influx (10 min) and a slower HDL protein internalization as determined by confocal microscopy and flow cytometry. Sphingomyelin kinetics overlapped that of apo AI, indicating that only cholesterol became dissociated from rHDL during internalization. rHDL apo AI internalization was scavenger receptor class B type I (SR-BI)-dependent, whereas HDL cholesterol influx was independent of SR-BI and was not completely inhibited by the presence of low-density lipoproteins (LDL). HDL sphingomyelin was fundamental for intercellular adhesion molecule-1 (ICAM-1) downregulation in HMEC-1. However, vascular cell adhesion protein-1 (VCAM-1) was not inhibited by rHDL, suggesting that components such as apolipoproteins other than apo AI participate in HDL’s regulation of this adhesion molecule. rHDL also induced endothelial nitric oxide synthase eNOS S1177 phosphorylation in HMEC-1 but only when the particle contained sphingomyelin. In conclusion, the internalization of HDL implies the dissociation of lipoprotein components and a SR-BI-independent fast delivery of cholesterol to endothelial cells. HDL internalization had functional implications that were mainly dependent on sphingomyelin. These results suggest a new role of HDL as lipid vectors to the cells, which could be congruent with the antiatherogenic properties of these lipoproteins.

Cell-based therapy has become a resource for the treatment of cardiovascular diseases; however, there are some conundrums to achieve. In vitro cardiomyocyte generation could be a solution for scaling options in clinical applications. Variability on cardiac differentiation in previously reported studies from adipose tissue-derived mesenchymal stem cells (ASCs) and the lack of measuring of the cardiomyocyte differentiation efficiency motivate the present study. Here, we improved the ASC-derived cardiomyocyte-like cell differentiation efficiency with a directed cardiomyocyte differentiation protocol: BMP-4 + VEGF (days 0-4) followed by a methylcellulose-based medium with cytokines (IL-6 and IL-3) (days 5-21). Cultures treated with the directed cardiomyocyte differentiation protocol showed cardiac-like cells and “rosette-like structures” from day 7. The percentage of cardiac troponin T- (cTnT-) positive cells was evaluated by flow cytometry to assess the cardiomyocyte differentiation efficiency in a quantitative manner. ASCs treated with the directed cardiomyocyte differentiation protocol obtained a differentiation efficiency of up to 44.03% (39.96%±3.78) at day 15 without any enrichment step. Also, at day 21 we observed by immunofluorescence the positive expression of early, late, and cardiac maturation differentiation markers (Gata-4, cTnT, cardiac myosin heavy chain (MyH), and the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCa2)) in cultures treated with the directed cardiomyocyte differentiation protocol. Unlike other protocols, the use of critical factors of embryonic cardiomyogenesis coupled with a methylcellulose-based medium containing previously reported cardiogenic cytokines (IL-6 and IL-3) seems to be favorable for in vitro cardiomyocyte generation. This novel efficient culture protocol makes ASC-derived cardiac differentiation more efficient. Further investigation is needed to identify an ASC-derived cardiomyocyte surface marker for cardiac enrichment.

In healthy conditions and cardiovascular diseases, the most abundant microparticles (MPs) in the bloodstream are those of platelet origin, but the direct effect of these microparticles on endothelial activation is poorly understood. The objective of this paper is to measure endothelial cell activation, as evaluated by the expression of the adhesion molecules E-selectin, VCAM-1, ICAM-1, and PECAM-1 in endothelial cell line HMEC-1 when stimulated with MPs produced by platelets stimulated in vitro with thrombin (TH), adenosine diphosphate (ADP), calcium ionophore (ICa), N-acetylglucosamine (NAcGlc), and without any stimulus. Platelets from healthy individuals induced the formation of MPs with different agonists. The results from the determination of the phenotype of the MPs showed that the expression of GPIIb/IIIa was significant, with median fold changes of TH = 2.2, ADP = 5.2, Ica = 7.0, and NAcGlc = 10.0. However, in HMEC-1 cells, the expression of adhesion molecules stimulated with MPs had a median change slightly higher for E-Sel expression (ranging from 1.4 to 4.2) and ICAM-1 expression (range 2.2 to 3.0), especially VCAM-1 expression (ranging from 15 to 18.8), all of which were significant. For PECAM-1, only stimulation with ICa (1.5) was significant, demonstrating that MPs elicit stimulus-dependent responses in endothelial cells. Platelet-derived MPs may have a potential role in modulating inflammation and other endothelial functions.

The potential transient vanilloid receptor type 1 (TRPV1) plays important functional roles in the vascular system. In the present study, we explored the role of the TRPV1 in the production of nitric oxide (NO), biopterines (BH4 and BH2), cyclic guanosine monophosphate (cGMP), malondialdehyde (MDA), phosphodiesterase‐3 (PDE‐3), total antioxidant capacity (TAC), and calcitonin gene‐related peptide (CGRP) in the rat aorta. Wistar rats were divided into four groups: (1) control, (2) capsaicin (CS, 20 mg/kg), (3) capsazepine (CZ, 24 mg/kg), and (4) CZ + CS. Treatments were applied daily for 4 days before removing the thoracic aortas for testing of aortic tissue and endothelial cells. TRPV1 activation produced increases in BH4 14%, cGMP 25%, NO 29%, and TAC 59.2% in comparison to the controls. BH2 and MDA increased with CZ. CGRP shows a tendency to decrease with CZ. The analysis by immunocytochemistry confirmed that the TRPV1 is present in aortic endothelial cells. Aortic endothelial cells were obtained from healthy rats and cultured to directly explore the effects of CS and CZ. The activation of the TRPV1 (CS 30 μM) produced increases in BH4 17%, NO 36.6%, TAC 56.3%, and CGRP 65%, when compared to controls. BH2 decreased with CZ + CS. CS effects were diminished by CZ in cells and in the tissue. We conclude that the TRPV1 is a structure present in the membrane of aortic endothelial cells and that it participates in the production of NO. The importance of the TRPV1 should be considered in vascular reactivity studies.

Fresh frozen plasma (FFP) administration may increase the risk of nosocomial infections in parallel with the development of immune modulation. This could be driven by soluble mediators, possibly influencing the in vitro activation of human U937 monocyte cells, in a manner dependent on the age of the donors. FFP donors were stratified into groups of 19–30 years, 31–40 years or 41–50 years, and U937 cells were cultured with FFP (alone or plus lipopolysaccharide—LPS) for 24 h. Both in FFP and supernatants, TNF, IL-1β, IL-6, and IL-10 levels were measured by ELISA. Additionally, CD11B, TLR2, and CASP3 gene expression were measured by qtPCR in U937 cells. Total phagocytic activity was also assayed. Elevated IL-10, but low TNF and IL-1β levels were measured in FFP from individuals aged 19–40 years, whereas in individuals aged 41–50 years FFP were characterized by equalized TNF and IL-10 levels. Elevated IL-6 levels were found in all FFP samples, especially in those from the oldest individuals. FFP stimulation was associated with striking modifications in cytokine production in an age-dependent way. Exposure to FFP attenuates the response to LPS. TLR2 and CD11B expression were enhanced regardless of the age of plasma donors, although CASP3 expression was increased only when FFP from individuals aged 19–40 years were tested. Phagocytosis decreased after exposure to FFP regardless of donor age. Our results suggest that soluble mediators in FFP may modulate the functioning of monocytes. Interestingly, this effect appears to be partially influenced by the age of donors.