Balbiani body of basal insects is potentially involved in multiplication and selective elimination of mitochondria.

Oocytes of both vertebrates and invertebrates often contain an intricate organelle assemblage, termed the Balbiani body (Bb). It has previously been suggested that this assemblage is involved in the delivery of organelles and macromolecules to the germ plasm, formation of oocyte reserve materials, and transfer of mitochondria to the next generation. To gain further insight into the function of the Bb, we performed a series of analyses and experiments, including computer-aided 3-dimensional reconstructions, detection of DNA (mtDNA) synthesis as well as immunolocalization studies. We showed that in orthopteran Meconema meridionale, the Bb comprises a network of mitochondria and perinuclear nuage aggregations. As oogenesis progresses, the network expands filling almost entire ooplasm, then partitions into several smaller entities, termed micro-networks, and ultimately into individual mitochondria. As in somatic cells, this process involves microfilaments and elements of endoplasmic reticulum. We showed also that at least some of the individual mitochondria are surrounded by phagophores and eliminated via mitophagy. These findings support the idea that the Bb is implicated in the multiplication and selective elimination of (defective) mitochondria and therefore may participate in the transfer of undamaged (healthy) mitochondria to the next generation.

Morphology and ultrastructure of the Balbiani body in the oocytes of closely related bush cricket species. Shared features reveal important aspect of functioning.

Balbiani bodies (Bbs) are female germline-specific organelle assemblages usually composed of mitochondria, Golgi complexes, elements of endoplasmic reticulum and accumulations of fine granular material, termed the nuage. Here we present results of morphological and ultrastructural analysis of the Bb of four bush crickets nested in four subfamilies of the family Tettigonidae. This study has revealed that Bbs of closely related species (belonging to the defined evolutionary line) are morphologically rather different. In two species (Meconema meridionale and Pholidoptera griseoaptera) the Bb has the form of a hollow hemisphere that covers a part of the germinal vesicle surface. In contrast, the Bb of Conocephalus fuscus and Leptophyes albovittata is less distinct and surrounds the whole or the majority of the germinal vesicle surface. Aside from this difference, the Bbs of all four studied species are built of identical sets of organelles and, most importantly, share one significant feature: close association of mitochondria and nuage accumulations. We show additionally that mitochondria remaining in direct contact with the nuage are characterized by distinct morphologies e.g. elongated, dumbbell shaped or bifurcated. In the light of our results and literature survey, the ancestral function of the Bb is discussed.

The Balbiani body: morphogenesis and functioning in the oocytes of vertebrates and invertebrates / Morfogeneza i rola ciala Balbianiego w oocytach kregowców i bezkregowców.

The Balbiani body is an organelle assemblage (termed sometimes a super-organelle) characteristic for the developing oocytes of almost all investigated animal species. In the vast majority of species, this complex resides next to the germinal vesicle and comprises such organelles as mitochondria, elements of endoplasmic reticulum, Golgi complexes as well as accumulations of nuage material. Comparative analyses have shown that the Balbiani bodies, even in closely related organisms, are often morphologically different. The differences concern not only the composition of this assemblage but also mutual relations between its components. So far, it has been found that the Balbiani body is implicated in several cellular processes undergoing in female germline cells. Most importantly this organelle complex is responsible for the delivery and localization of certain macromolecules and organelles to specific regions of the ooplasm (oocyte cytoplasm), as well as in the transfer of mitochondria to the zygote, i.e. to the next generation. Moreover, it has been shown recently that at least in some species the Balbiani body participates in the elimination of nonfunctional, damaged mitochondria from the developing oocytes and egg cells.

Morphogenesis of the Balbiani body in developing oocytes of an orthopteran, Metrioptera brachyptera, and multiplication of female germline mitochondria.

Balbiani body (Bb) is a female germline specific organelle complex. Although the morphology and morphogenesis of the Bb have been analyzed in numerous vertebrate and invertebrate species, the role and ultimate fate of this organelle assemblage are still under debate. As a result, various functions have been attributed to the Bb in given animal lineages or even species. Our analyses showed that in the bush cricket, Metrioptera brachyptera, the Bb is an elaborate and highly dynamic structure positioned at one side of the oocyte nucleus. It forms in early previtellogenic oocytes and consists of two compartments: perinuclear and cytoplasmic. In the cytoplasmic compartment, characteristic complexes of nuage and polymorphous mitochondria are present. Computer-aided 3D reconstructions revealed that mitochondria clustered around neighboring nuage accumulations remain in a physical contact and form an extensive, though dispersed network. As oogenesis progresses, nuage/mitochondria complexes are partitioned into progressively smaller entities that become separated from each other. Concurrently, the mitochondrial network splits into small individual mitochondria populating the whole ooplasm. Immunohistochemical analysis showed that the latter process involves dynamin-related protein 1 (Drp1). Collectively, our findings suggest that in basal insect species, the Bb might be responsible for the selection as well as multiplication of the oocyte mitochondria.

Transmission of Functional, Wild-Type Mitochondria and the Fittest mtDNA to the Next Generation: Bottleneck Phenomenon, Balbiani Body, and Mitophagy.

The most important role of mitochondria is to supply cells with metabolic energy in the form of adenosine triphosphate (ATP). As synthesis of ATP molecules is accompanied by the generation of reactive oxygen species (ROS), mitochondrial DNA (mtDNA) is highly vulnerable to impairment and, consequently, accumulation of deleterious mutations. In most animals, mitochondria are transmitted to the next generation maternally, i.e., exclusively from female germline cells (oocytes and eggs). It has been suggested, in this context, that a specialized mechanism must operate in the developing oocytes enabling escape from the impairment and subsequent transmission of accurate (devoid of mutations) mtDNA from one generation to the next. Literature survey suggest that two distinct and irreplaceable pathways of mitochondria transmission may be operational in various animal lineages. In some taxa, the mitochondria are apparently selected: functional mitochondria with high inner membrane potential are transferred to the cells of the embryo, whereas those with low membrane potential (overloaded with mutations in mtDNA) are eliminated by mitophagy. In other species, the respiratory activity of germline mitochondria is suppressed and ROS production alleviated leading to the same final effect, i.e., transmission of undamaged mitochondria to offspring, via an entirely different route.

Morphogenesis of the ovarian follicular epithelium during initial stages of embryogenesis of the viviparous earwig, Hemimerus talpoides.

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended "postfertilization" activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.

Viviparity in the dermapteran Arixenia esau: respiration inside mother's body requires both maternal and larval contribution.

Earwigs (Dermaptera) use different strategies to increase their reproductive success. Most species lay eggs; however, viviparity of the matrotrophic type has been reported in two groups: Hemimeridae and Arixeniidae. In Arixeniidae, offspring develop in two separate places: inside an ovary (the intraovarian phase) and within a uterus (the intrauterine phase). Both morphological and physiological aspects of viviparity in Arixeniidae have begun to be unraveled only recently. Here, we characterize how the first instar larvae of Arixenia esau, developing inside the mother's reproductive system, manage respiration and gas exchange. Using modern light and electron microscopy techniques as well as immunological approach, we provide a detailed account of the maternal and larval tissue interactions during the intrauterine development. We demonstrate that respiration in the Arixenia first instar larvae relies on the extensive tracheal system of the mother as well as a respiratory pigment (hemocyanin) present within the body cavity of the larvae. Our results indicate that the larval fat body tissue is the likely place of the hemocyanin synthesis. Our study shows that characteristic cone-shaped lobes of the outgrowths located on the larval abdomen are a part of a placenta-like organ and mediate the gas exchange between the maternal and larval organisms. Based on the obtained results, we propose that Arixenia esau evolved a unique biphasic system supporting respiration of the first instar larvae during their development inside the mother's reproductive tract.

Insights into the role of estrogen-related receptors α, ß and γ in tumor Leydig cells.

In this study, we demonstrate, for the first time, estrogen-related receptor (ERR) regulation of the physiological and biochemical status of testicular tumor Leydig cells. In a mouse tumor Leydig cells, ERRs (α, ß, and γ) were silenced via siRNA. Cell morphology and cell physiology (proliferation and observation of monolayer formation) were performed by inverted phase-contrast microscope. Leydig cell functional markers (steroid receptors and signaling molecules) were examined by immunofluorescence and Western blotting. Additionally, progesterone secretion was assessed. Mitochondrial mass and membrane potential were analyzed by flow-cytometry while cGMP and Ca2+ concentrations were analyzed using immunoenzymatic and colorimetric assays, respectively. These results revealed, ERRs indirectly regulate Leydig cell proliferation while ERRα and ß affect cell monolayer formation. ERRs interact with canonical and membrane estrogen receptors (ERα, ERß, and GPER), androgen receptor, metalloproteinase (MMP 9), protein kinase A (PKA), extracellular-regulated kinase (ERK), and neurogenic locus notch homolog protein 2 (Notch2). Depending on the type of ERR knocked down, coupled with estradiol treatment, changes in progesterone concentration and cGMP and Ca2+ concentrations constitute a microenvironment that may effect tumor Leydig cell characteristics. ERRs should be considered important factors in developing of innovating approaches that target pathological processes of testicular Leydig cells.

Induced Pluripotent Stem Cell (iPSC)-Derived Extracellular Vesicles Are Safer and More Effective for Cardiac Repair Than iPSCs.

RATIONALE: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs. OBJECTIVE: To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo. METHODS AND RESULTS: Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV-treated mice exhibited improved left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe. CONCLUSIONS: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.

Polylactide- and polycaprolactone-based substrates enhance angiogenic potential of human umbilical cord-derived mesenchymal stem cells in vitro - implications for cardiovascular repair.

Recent approaches in tissue regeneration focus on combining innovative achievements of stem cell biology and biomaterial sciences to develop novel therapeutic strategies for patients. Growing recent evidence indicates that mesenchymal stem cells harvested from human umbilical cord Wharton's jelly (hUC-MSCs) are a new valuable source of cells for autologous as well as allogeneic therapies in humans. hUC-MSCs are multipotent, highly proliferating cells with prominent immunoregulatory activity. In this study, we evaluated the impact of widely used FDA approved poly(α-esters) including polylactide (PLA) and polycaprolactone (PCL) on selected biological properties of hUC-MSCs in vitro. We found that both polymers can be used as non-toxic substrates for ex vivo propagation of hUC-MSCs as shown by no major impact on cell proliferation or viability. Moreover, PCL significantly enhanced the migratory capacity of hUC-MSCs. Importantly, genetic analysis indicated that both polymers promoted the angiogenic differentiation potential of hUC-MSCs with no additional chemical stimulation. These results indicate that PLA and PCL enhance selected biological properties of hUC-MSCs essential for their regenerative capacity including migratory and proangiogenic potential, which are required for effective vascular repair in vivo. Thus, PLA and PCL-based scaffolds combined with hUC-MSCs may be potentially employed as future novel grafts in tissue regeneration such as blood vessel reconstruction.

Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles.

Growing evidence indicates that intracellular signaling mediated by extracellular vesicles (EVs) released by stem cells plays a considerable role in triggering the regenerative program upon transplantation. EVs from umbilical cord mesenchymal stem cells (UC-MSC-EVs) have been shown to enhance tissue repair in animal models. However, translating such results into clinical practice requires optimized EV collection procedures devoid of animal-originating agents. Thus, in this study, we analyzed the influence of xeno-free expansion media on biological properties of UC-MSCs and UC-MSC-EVs for future applications in cardiac repair in humans. Our results show that proliferation, differentiation, phenotype stability, and cytokine secretion by UC-MSCs vary depending on the type of xeno-free media. Importantly, we found distinct molecular and functional properties of xeno-free UC-MSC-EVs including enhanced cardiomyogenic and angiogenic potential impacting on target cells, which may be explained by elevated concentration of several pro-cardiogenic and pro-angiogenic microRNA (miRNAs) present in the EVs. Our data also suggest predominantly low immunogenic capacity of certain xeno-free UC-MSC-EVs reflected by their inhibitory effect on proliferation of immune cells in vitro. Summarizing, conscious selection of cell culture conditions is required to harvest UC-MSC-EVs with the optimal desired properties including enhanced cardiac and angiogenic capacity, suitable for tissue regeneration. KEY MESSAGE: Type of xeno-free media influences biological properties of UC-MSCs in vitro. Certain xeno-free media promote proliferation and differentiation ability of UC-MSCs. EVs collected from xeno-free cultures of UC-MSCs are biologically active. Xeno-free UC-MSC-EVs enhance cardiac and angiogenic potential of target cells. Type of xeno-free media determines immunomodulatory effects mediated by UC-MSC-EVs.

Monocyte Chemoattractant Protein-Induced Protein 1 (MCPIP1) Enhances Angiogenic and Cardiomyogenic Potential of Murine Bone Marrow-Derived Mesenchymal Stem Cells.

The current evidence suggests that beneficial effects of mesenchymal stem cells (MSCs) toward myocardial repair are largely due to paracrine actions of several factors. Although Monocyte chemoattractant protein-induced protein 1 (MCPIP1) is involved in the regulation of inflammatory response, apoptosis and angiogenesis, whether MCPIP1 plays any role in stem cell-induced cardiac repair has never been examined. By employing retroviral (RV)-transduced overexpression of MCPIP1, we investigated the impact of MCPIP1 on viability, apoptosis, proliferation, metabolic activity, proteome, secretome and differentiation capacity of murine bone marrow (BM) - derived MSCs. MCPIP1 overexpression enhanced angiogenic and cardiac differentiation of MSCs compared with controls as indicated by elevated expression of genes accompanying angiogenesis and cardiomyogenesis in vitro. The proangiogenic activity of MCPIP1-overexpressing MSCs (MCPIP1-MSCs) was also confirmed by increased capillary-like structure formation under several culture conditions. This increase in differentiation capacity was associated with decreased proliferation of MCPIP1-MSCs when compared with controls. MCPIP1-MSCs also expressed increased levels of proteins involved in angiogenesis, autophagy, and induction of differentiation, but not adverse inflammatory agents. We conclude that MCPIP1 enhances endothelial and cardiac differentiation of MSCs. Thus, modulating MCPIP1 expression may be a novel approach useful for enhancing the immune-regulatory, anti-apoptotic, anti-inflammatory and regenerative capacity of BM-derived MSCs for myocardial repair and regeneration of ischemic tissues.

Human Induced Pluripotent Stem Cell-Derived Microvesicles Transmit RNAs and Proteins to Recipient Mature Heart Cells Modulating Cell Fate and Behavior.

Microvesicles (MVs) are membrane-enclosed cytoplasmic fragments released by normal and activated cells that have been described as important mediators of cell-to-cell communication. Although the ability of human induced pluripotent stem cells (hiPSCs) to participate in tissue repair is being increasingly recognized, the use of hiPSC-derived MVs (hiPSC-MVs) in this regard remains unknown. Accordingly, we investigated the ability of hiPSC-MVs to transfer bioactive molecules including mRNA, microRNA (miRNA), and proteins to mature target cells such as cardiac mesenchymal stromal cells (cMSCs), and we next analyzed effects of hiPSC-MVs on fate and behavior of such target cells. The results show that hiPSC-MVs derived from integration-free hiPSCs cultured under serum-free and feeder-free conditions are rich in mRNA, miRNA, and proteins originated from parent cells; however, the levels of expression vary between donor cells and MVs. Importantly, we found that transfer of hiPSC components by hiPSC-MVs impacted on transcriptome and proteomic profiles of target cells as well as exerted proliferative and protective effects on cMSCs, and enhanced their cardiac and endothelial differentiation potential. hiPSC-MVs also transferred exogenous transcripts from genetically modified hiPSCs that opens new perspectives for future strategies to enhance MV content. We conclude that hiPSC-MVs are effective vehicles for transferring iPSC attributes to adult somatic cells, and hiPSC-MV-mediated horizontal transfer of RNAs and proteins to injured tissues may be used for therapeutic tissue repair. In this study, for the first time, we propose a new concept of use of hiPSCs as a source of safe acellular bioactive derivatives for tissue regeneration.

MET receptor is a potential therapeutic target in high grade cervical cancer.

Cervical cancer is one of the leading causes of death among women suffering from tumors. Current treatment options are insufficient. Here, we investigated the MET receptor as a potential molecular target in advanced cervical cancer. Downregulation of MET receptor expression via RNA interference in different cervical carcinoma cell lines dramatically decreased tumor growth and forced tumor differentiation in vivo. MET receptor silencing also led to a dramatic decrease in cell size and a decrease in proliferation rate under normal and stress conditions. MET receptor downregulation also resulted in decreased cyclin D1 and c-myc levels but did not increase apoptosis. Subsequent experiments showed that downregulation of the MET receptor decreased the expression of a key regulator of the epithelial-to-mesenchymal transition, SLUG. and increased the expression of E-cadherin, a hallmark of the epithelial phenotype. Moreover, MET downregulation impairs expression and signaling of CXCR4 receptor, responsible for invasive phenotype. Taken together, our results strongly suggest that the MET receptor influences the oncogenic properties of cervical carcinoma cells in vitro and in vivo. These findings highlight a unique role of the MET receptor in cervical carcinoma cells and indicate the MET receptor as a potential therapeutic target for advanced cervical carcinoma.

The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

Downregulation of the CXCR4 receptor inhibits cervical carcinoma metastatic behavior in vitro and in vivo.

Cervical carcinoma is frequently diagnosed among women, particularly in low and middle income countries. In this study, we investigated the role of the SDF-1/CXCR4 axis during cervical carcinoma growth and progression in vitro and in vivo. Downregulation of CXCR4 receptor using an RNA interference system led to almost complete inhibition of the receptor expression, activation and function. CXCR4 receptor silencing led to decreased ability to signal, to induce migration and to form holoclone-like colonies, with no influence on viability/proliferation of the cells. CXCR4-deficient cells had also significantly lower levels of MMP-9. Interestingly, downregulation of CXCR4 expression resulted in reduced tumor growth in vivo. Tumors generated by CXCR4-deficient cells had also lower expression of the proliferation marker Ki­67 and decreased ability to engraft into lungs and spleen. Taken together, our results indicate that CXCR4 receptor may play an important role during cervical carcinoma invasion. In our study CXCR4 influenced invasive properties of cervical carcinoma cells both in vitro and in vivo.

17AEP-GA, an HSP90 antagonist, is a potent inhibitor of glioblastoma cell proliferation, survival, migration and invasion.

Glioblastoma multiforme (GBM) is the most frequent and the most malignant human brain tumor. The expression of receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) is strongly increased in GBM, where they promote tumor proliferation, cell survival, migration, invasion and angiogenesis. We used geldanamycins (GAs) (inhibitors of HSP90) in order to block glioblastoma growth and HGF-dependent cell migration and invasion. The effect of GAs on three GBM cell lines was tested and we found their antiproliferative effect on tumor cells. The maximum level of inhibition reached 70%. After treatment with GAs, cells also became apoptotic as determined by Annexin V-positive staining and activation of the caspase-3 pathway. We examined the expression and activity of the MET receptor on GBM cell lines and we observed phosphorylation of AKT and MAPK after HGF stimulation by western blot analysis. Since GBM cells express high level of MET receptor and were shown to respond to HGF by increased motility we tested if GAs could negatively affect GBM cell movement. In our study, we found that GAs inhibited the chemotaxis of glioblastoma cells toward the hepatocyte growth factor gradient. The GAs also blocked migration of tumor cells through a Matrigel layer in invasion assays. The strongest inhibitory effect was observed for GA and its analog, 17AEP-GA. Based on our results, GAs, particularly 17AEP-GA, could be considered as a new potential agent to treat glioblastoma multiforme.

Endothelial microparticle formation in moderate concentrations of homocysteine and methionine in vitro.

Microparticles (MPs) are small membrane vesicles released by stimulated or apoptotic cells, including the endothelium. Hyperhomocysteinemia (HHcy) is a blood disorder characterized by an increase in the plasma concentrations of total homocysteine (Hcy). The plasma Hcy level is determined by environmental factors (dietary habits, i.e. the intake of folic acid, FA) and genetic factors (N (5),N (10)-methylenetetrahydro-folate reductase, MTHFR, polymorphism 677C>T). To evaluate whether moderate Hcy concentrations induce endothelial MP formation, the role of FA supplementation and the influence of MTHFR polymorphism were analysed. Human umbilical vein endothelial cells (HUVEC) were treated in vitro with 50 µM of Hcy and methionine (Met). The MP number and apoptotic phenotype were analyzed using flow cytometry. Increasing doses of FA (5, 15 and 50 µM) were used to reduce the HHcy effect. The MTHFR 677C>T polymorphism was determined. HUVEC stimulated by Hcy produced significantly more MPs than HUVEC under the control conditions: 3,551 ± 620 vs 2,270 ± 657 kMP (p = 0.02). Supplementation with FA at concentrations of 5, 15 and 50 µM reduced the MP count in the cell culture supernatant to 345 ± 332, 873 ± 329, and 688 ± 453 kMP, respectively (p = 0.03). MTHFR 677C>T heterozygosity was associated with a significant increase in MP formation after stimulation with Hcy compared to the control conditions: 3,617 ± 152 vs 1,518 ± 343 kMP (p = 0.02). Furthermore, the MTHFR genotype altered MP formation after Met loading. On average, 24% of the entire MP population was apoptotic (annexin V-positive). Endothelial function impairment due to HHcy is related to MP shedding, which may involve platelets and other blood and vascular cells. MP shedding is a physiological response to moderate HHcy.