Author Correction: Conversion of human fibroblasts to angioblast-like progenitor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Conversion of human fibroblasts to angioblast-like progenitor cells.

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.

Interactions between hypertension and inflammatory tone and the effect on blood pressure and outcomes in patients with COVID-19.

Arterial hypertension represented one of the most common comorbidities in patients with COVID-19. However, the impact of hypertension on outcome in COVID-19 patients is not clear. Close connections between inflammation and blood pressure (BP) have been described, and inflammation plays a key role in the outcome for patients with COVID-19. Whether hypertension impairs the relationship between inflammation, BP, and outcomes in this context is not known. The aim of this study was to examine the effects of the interactions between inflammation and hypertension status on BP and clinical outcome in patients hospitalized with COVID-19. We designed a retrospective study in 129 patients hospitalized with COVID-19 at Toulouse University Hospital. The hospital outcome was admission to the intensive care unit or death. The inflammatory markers were blood C-reactive protein level (CRP), neutrophil to lymphocyte, and platelet to lymphocyte ratios. We identified strong correlations between CRP (P < .01) and the other inflammatory markers recorded on admission (P < .001) with mean BP within 3 days after admission in normotensive patients, whereas these correlations were absent in patients with hypertension. Also, we observed after multivariate adjustment (P < .05) that CRP level predicted a worse prognosis in hypertensive patients (relative risk 2.52; 95% confidence intervals [1.03- 6.17]; P = .04), whereas CRP was not predictive of outcome in patients without hypertension. In conclusion, the study revealed that in COVID-19 patients, hypertension impairs the relationship between inflammation and BP and interacts with inflammation to affect prognosis. These findings provide insights that could explain the relationship between hypertension and outcomes in COVID-19 patients.

PtdIns5P: a little phosphoinositide with big functions?

Phosphoinositides are minor constituents of cell membranes playing a critical role in the regulation of many cellular functions. Recent discoveries indicate that mutations in several phosphoinositide kinases and phosphatases generate imbalances in the levels of phosphoinositides, thereby leading to the development of human diseases. Although the roles of phosphoinositide 3-kinase products and PtdIns(4,5)P2 were largely studied these last years, the potential role of phosphatidylinositol monophosphates as direct signalling molecules is just emerging. PtdIns5P, the least characterized phosphoinositide, appears to be a new player in cell regulation. This review will summarize the current knowledge on the mechanisms of synthesis and degradation of PtdIns5P as well as its potential roles.

Emerging roles of phosphatidylinositol monophosphates in cellular signaling and trafficking.

The phosphoinositide metabolism that is highly controlled by a set of kinases, phosphatases and phospholipases leads to the production of several second messengers playing critical roles in intracellular signal transduction mechanisms. Recent discoveries have unraveled unexpected roles for the three phosphatidylinositol monophosphates, PtdIns(3)P, PtdIns(4)P and PtdIns(5)P, that appear now as important lipid messengers able to specifically interact with proteins. The formation of functionally distinct and independently regulated pools of phosphatidylinositol monophosphates probably contributes to the specificity of the interactions with their targets. The relative enrichment of organelles in a particular species of phosphoinositides (i.e. PtdIns(3)P in endosomes, PtdIns(4)P in Golgi and PtdIns(4,5)P2 in plasma membrane) suggests the notion of lipid-defined organelle identity. PtdIns(3)P is now clearly involved in vesicular trafficking by interaction with a set of FYVE domain-containing proteins both in yeast and in mammals. PtdIns(4)P, which until now was only considered as a precursor for PtdIns(4,5)P2, appears as a regulator on its own, by recruiting a set of proteins to the trans-Golgi network. PtdIns(5)P, the most recently discovered inositol lipid, is also emerging as a potentially important signaling molecule.

Phosphoinositide signaling disorders in human diseases.

Phosphoinositides (PIs) play an essential role in diverse cellular functions. Their intracellular level is strictly regulated by specific PI kinases, phosphatases and phospholipases. Recent discoveries indicate that dysfunctions in the control of their level often lead to pathologies. This review will focus on some human diseases whose etiologies involve PI-metabolizing enzymes. The role of PTEN (phosphatase and tensin homolog deleted on chromosome ten) in cancer, the impact of the Src homology 2-containing inositol-5-phosphatase phosphatases in acute myeloid leukemia or diabetes, the involvement of myotubularin family members in genetic diseases and the implication of OCRL1 in Lowe syndrome will be emphasized. We will also review how some bacterial pathogens have evolved strategies to specifically manipulate the host cell PI metabolism to efficiently infect them.

Regulator of G-protein signaling 18 controls both platelet generation and function.

RGS18 is a myeloerythroid lineage-specific regulator of G-protein signaling, highly expressed in megakaryocytes (MKs) and platelets. In the present study, we describe the first generation of a RGS18 knockout mouse model (RGS18-/-). Interesting phenotypic differences between RGS18-/- and wild-type (WT) mice were identified, and show that RGS18 plays a significant role in both platelet generation and function. RGS18 deficiency produced a gain of function phenotype in platelets. In resting platelets, the level of CD62P expression was increased in RGS18-/- mice. This increase correlated with a higher level of plasmatic serotonin concentration. RGS18-/- platelets displayed a higher sensitivity to activation in vitro. RGS18 deficiency markedly increased thrombus formation in vivo. In addition, RGS18-/- mice presented a mild thrombocytopenia, accompanied with a marked deficit in MK number in the bone marrow. Analysis of MK maturation in vitro and in vivo revealed a defective megakaryopoiesis in RGS18-/- mice, with a lower bone marrow content of only the most committed MK precursors. Finally, RGS18 deficiency was correlated to a defect of platelet recovery in vivo under acute conditions of thrombocytopenia. Thus, we highlight a role for RGS18 in platelet generation and function, and provide additional insights into the physiology of RGS18.

Methods for genetic modification of megakaryocytes and platelets.

During recent decades there have been major advances in the fields of thrombosis and haemostasis, in part through development of powerful molecular and genetic technologies. Nevertheless, genetic modification of megakaryocytes and generation of mutant platelets in vitro remains a highly specialized area of research. Developments are hampered by the low frequency of megakaryocytes and their progenitors, a poor efficiency of transfection and a lack of understanding with regard to the mechanism by which megakaryocytes release platelets. Current methods used in the generation of genetically modified megakaryocytes and platelets include mutant mouse models, cell line studies and use of viruses to transform primary megakaryocytes or haematopoietic precursor cells. This review summarizes the advantages, limitations and technical challenges of such methods, with a particular focus on recent successes and advances in this rapidly progressing field including the potential for use in gene therapy for treatment of patients with platelet disorders.

A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice.

During thrombopoiesis, maturing megakaryocytes (MKs) migrate within the complex bone marrow stromal microenvironment from the proliferative osteoblastic niche to the capillary-rich vascular niche where proplatelet formation and platelet release occurs. This physiologic process involves proliferation, differentiation, migration, and maturation of MKs before platelet production occurs. In this study, we report a role for the glycoprotein PECAM-1 in thrombopoiesis. We show that following induced thrombocytopenia, recovery of the peripheral platelet count is impaired in PECAM-1-deficient mice. Whereas MK maturation, proplatelet formation, and platelet production under in vitro conditions were unaffected, we identified a migration defect in PECAM-1-deficient MKs in response to a gradient of stromal cell-derived factor 1 (SDF1), a major chemokine regulating MK migration within the bone marrow. This defect could be explained by defective PECAM-1(-/-) MK polarization of the SDF1 receptor CXCR4 and an increase in adhesion to immobilized bone marrow matrix proteins that can be explained by an increase in integrin activation. The defect of migration and polarization was confirmed in vivo with demonstration of altered spatial localization of MKs within the bone marrow in PECAM-1-deficient mice, following immune-induced thrombocytopenia. This study identifies a novel role for PECAM-1 in regulating MK migration and thrombopoiesis.

PtdIns5P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection.

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns5P). We show that PtdIns5P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns5P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns5P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns5P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns5P production that plays an important role in host cell responses such as survival.

Production of phosphatidylinositol 5-phosphate by the phosphoinositide 3-phosphatase myotubularin in mammalian cells.

MTM1, the gene encoding myotubularin (MTM1), is mutated in the X-linked myotubular myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of myotubular myopathy.