Antoine A.F. de Vries

The Genome Organization of the Nidovirales: Similarities and Differences between Arteri-, Toro-, and Coronaviruses

n Abstractn n Viruses in the families Arteriviridae and Coronaviridae have enveloped virions which contain nonsegmented, positive-stranded RNA, but the constituent genera differ markedly in genetic complexity and virion structure. Nevertheless, there are striking resemblances among the viruses in the organization and expression of their genomes, and sequence conservation among the polymerase polyproteins strongly suggests that they have a common ancestry. On this basis, the International Committee on Taxonomy of Viruses recently established a new order, Nidovirales, to contain the two families. Here, the common traits and distinguishing features of the Nidovirales are reviewed.n n

Epicardial Cells of Human Adults Can Undergo an Epithelial‐to‐Mesenchymal Transition and Obtain Characteristics of Smooth Muscle Cells In Vitro

Myocardial and coronary development are both critically dependent on epicardial cells. During cardiomorphogenesis, a subset of epicardial cells undergoes an epithelial‐to‐mesenchymal transition (EMT) and invades the myocardium to differentiate into various cell types, including coronary smooth muscle cells and perivascular and cardiac interstitial fibroblasts. Our current knowledge of epicardial EMT and the ensuing epicardium‐derived cells (EPDCs) comes primarily from studies of chick and mouse embryonic development. Due to the absence of an in vitro culture system, very little is known about human EPDCs. Here, we report for the first time the establishment of cultures of primary epicardial cells from human adults and describe their immunophenotype, transcriptome, transducibility, and differentiation potential in vitro. Changes in morphology and β‐catenin staining pattern indicated that human epicardial cells spontaneously undergo EMT early during ex vivo culture. The surface antigen profile of the cells after EMT closely resembles that of subepithelial fibroblasts; however, only EPDCs express the cardiac marker genes GATA4 and cardiac troponin T. After infection with an adenovirus vector encoding the transcription factor myocardin or after treatment with transforming growth factor‐β1 or bone morphogenetic protein‐2, EPDCs obtain characteristics of smooth muscle cells. Moreover, EPDCs can undergo osteogenesis but fail to form adipocytes or endothelial cells in vitro. Cultured epicardial cells from human adults recapitulate at least part of the differentiation potential of their embryonic counterparts and represent an excellent model system to explore the biological properties and therapeutic potential of these cells.

Forced Alignment of Mesenchymal Stem Cells Undergoing Cardiomyogenic Differentiation Affects Functional Integration With Cardiomyocyte Cultures

Alignment of cardiomyocytes (CMCs) contributes to the anisotropic (direction-related) tissue structure of the heart, thereby facilitating efficient electrical and mechanical activation of the ventricles. This study aimed to investigate the effects of forced alignment of stem cells during cardiomyogenic differentiation on their functional integration with CMC cultures. Labeled neonatal rat (nr) mesenchymal stem cells (nrMSCs) were allowed to differentiate into functional heart muscle cells in different cell-alignment patterns during 10 days of coculture with nrCMCs. Development of functional cellular properties was assessed by measuring impulse transmission across these stem cells between 2 adjacent nrCMC fields, cultured onto microelectrode arrays and previously separated by a laser-dissected channel (230±10 &mgr;m) for nrMSC transplantation. Coatings in these channels were microabraded in a direction (1) parallel or (2) perpendicular to the channel or were (3) left unabraded to establish different cell patterns. Application of cells onto microabraded coatings resulted in anisotropic cell alignment within the channel. Application on unabraded coatings resulted in isotropic (random) alignment. After coculture, conduction across seeded nrMSCs occurred from day 1 (perpendicular and isotropic) or day 6 (parallel) onward. Conduction velocity across nrMSCs at day 10 was highest in the perpendicular (11±0.9 cm/sec; n=12), intermediate in the isotropic (7.1±1 cm/sec; n=11) and lowest in the parallel configuration (4.9±1 cm/sec; n=11) (P<0.01). nrCMCs and fibroblasts served as positive and negative control, respectively. Also, immunocytochemical analysis showed alignment-dependent increases in connexin 43 expression. In conclusion, forced alignment of nrMSCs undergoing cardiomyogenic differentiation affects the time course and degree of functional integration with surrounding cardiac tissue.

Structural protein requirements in equine arteritis virus assembly.

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP2b (previously named GS), GP3, GP4, and GP5 (previously named GL). Proteins N, M, and GP5 are major virion components, E occurs in virus particles in intermediate amounts, and GP4, GP3, and GP2b are minor structural proteins. The M and GP5 proteins occur in virus particles as disulfide-linked heterodimers while the GP4, GP3, and GP2b proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP2b, GP3, or GP4 proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP2b, GP3, and GP4 proteins into viral particles. EAV particles lacking GP2b, GP3, GP4, and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP2b/GP3/GP4 heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.

Highly Efficient Targeted Transduction of Undifferentiated Human Hematopoietic Cells by Adenoviral Vectors Displaying Fiber Knobs of Subgroup B

Human hematopoietic stem cells (HSCs) are poorly transduced by vectors based on adenovirus serotype 5 (Ad5). This is primarily due to the paucity of the coxsackievirus-Ad receptor on these cells. In an attempt to change the tropism of Ad5, we constructed a series of chimeric E1-deleted Ad5 vectors in which the shaft and knob of the capsid fibers were exchanged with those of other Ad serotypes. In all these vectors, the Ad E1 region was replaced by an expression cassette containing the cytomegalovirus immediate-early promoter and the gene for enhanced green fluorescent protein (GFP). Experiments performed in vitro showed an efficient transduction of umbilical cord blood (UCB) monocytes, granulocytes, and their precursors as well as the undifferentiated CD34(+) CD33(-) CD38(-) CD71(-) cells by Ad5 vectors carrying Ad subgroup B-specific fiber chimeras (Ad5FBs). In the latter subpopulation, which comprises less than 1% of the CD34(+) cells and is highly enriched with cells repopulating immunodeficient mice, more than 90% of the cells were GFP(+). Transduction by Ad5FBs of the less primitive fraction within UCB CD34(+) cells was significant lower. Actually, the transduction frequency and GFP level declined gradually with increased expression of the CD33, CD38, and CD71 antigens. Flow cytometric analysis of transduced UCB CD34(+) cells that were cultured for 5 days on an allogeneic human bone marrow stroma layer showed maintenance of the phenotypically defined HSCs at levels similar to those of control cultures. The latter finding indicates that neither the transduction procedure nor the high levels of GFP were toxic for these cells.

Forced Myocardin Expression Enhances the Therapeutic Effect of Human Mesenchymal Stem Cells After Transplantation in Ischemic Mouse Hearts

Human mesenchymal stem cells (hMSCs) have only a limited differentiation potential toward cardiomyocytes. Forced expression of the cardiomyogenic transcription factor myocardin may stimulate hMSCs to acquire a cardiomyogenic phenotype, thereby improving their possible therapeutic potential. hMSCs were transduced with green fluorescent protein (GFP) and myocardin (hMSCmyoc) or GFP and empty vector (hMSC). After coronary ligation in immune‐compromised NOD/scid mice, hMSCmyoc (n = 10), hMSC (n = 10), or medium only (n = 12) was injected into the infarct area. Sham‐operated mice (n = 12) were used to determine baseline characteristics. Left ventricular (LV) volumes and ejection fraction (EF) were serially (days 2 and 14) assessed using 9.4‐T magnetic resonance imaging. LV pressure‐volume measurements were performed at day 15, followed by histological evaluation. At day 2, no differences in infarct size, LV volumes, or EF were observed among the myocardial infarction groups. At day 14, left ventricular ejection fraction in both cell‐treated groups was preserved compared with the nontreated group; in addition, hMSCmyoc injection also reduced LV volumes compared with medium injection (p < .05). Furthermore, pressure‐volume measurements revealed a significantly better LV function after hMSCmyoc injection compared with hMSC treatment. Immunohistochemistry at day 15 demonstrated that the engraftment rate was higher in the hMSCmyoc group compared with the hMSC group (p < .05). Furthermore, these cells expressed a number of cardiomyocyte‐specific markers not observed in the hMSC group. After myocardial infarction, injection of hMSCmyoc improved LV function and limited LV remodeling, effects not observed after injection of hMSC. Furthermore, forced myocardin expression improved engraftment and induced a cardiomyocyte‐like phenotype hMSC differentiation.

Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus.

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP5 (previously named GL), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP2b (formerly designated GS), GP3, and GP4. Analysis of the appearance of the GP2b, GP3, and GP4 proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP2b and GP4 and heterotrimers of GP2b, GP3, and GP4. Shortly after their release from infected cells, virions contained mainly cystine-linked GP2b/GP4 heterodimers, which were subsequently converted into disulfide-bonded GP2b/GP3/GP4 trimers through the covalent recruitment of GP3. This process occurred faster at a higher pH but was arrested at 4°C. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N-ethylmaleimide inhibited the formation of disulfide-bonded GP2b/GP3/GP4 trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP3 with the cystine-linked GP2b/GP4 dimer in freshly released virions, nor did we observe higher-order structures of the GP2b/GP4 or GP2b/GP3/GP4 complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP2b/GP3/GP4 heterotrimers at 4°C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP2b/GP4 dimer and GP3 was also inferred from coexpression experiments showing that the presence of GP3 increased the electrophoretic mobility of the disulfide-bonded GP2b/GP4 dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.

Human Embryonic and Fetal Mesenchymal Stem Cells Differentiate toward Three Different Cardiac Lineages in Contrast to Their Adult Counterparts

Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.

Hematopoietic MicroRNA-126 Protects against Renal Ischemia/Reperfusion Injury by Promoting Vascular Integrity

Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin(-)/Sca-1(+)/cKit(+) hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin(-)/Sca-1(+)/cKit(+) cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin(-)/Sca-1(+)/cKit(+) cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.

Myogenic properties of human mesenchymal stem cells derived from three different sources.

Mesenchymal stem cells (MSCs) of mammals have been isolated from many tissues and are characterized by their aptitude to differentiate into bone, cartilage, and fat. Differentiation into cells of other lineages like skeletal muscle, tendon/ligament, nervous tissue, and epithelium has been attained with MSCs derived from some tissues. Whether such abilities are shared by MSCs of all tissues is unknown. We therefore compared for three human donors the myogenic properties of MSCs from adipose tissue (AT), bone marrow (BM), and synovial membrane (SM). Our data show that human MSCs derived from the three tissues differ in phenotype, proliferation capacity, and differentiation potential. The division rate of AT-derived MSCs (AT-MSCs) was distinctly higher than that of MSCs from the other two tissue sources. In addition, clear donor-specific differences in the long-term maintenance of MSC proliferation ability were observed. Although similar in their in vitro fusogenic capacity with murine myoblasts, MSCs of the three sources contributed to a different extent to skeletal muscle regeneration in vivo. Transplanting human AT-, BM-, or SM-MSCs previously transduced with a lentiviral vector encoding β-galactosidase into cardiotoxin-damaged tibialis anterior muscles (TAMs) of immunodeficient mice revealed that at 30 days after treatment the frequency of hybrid myofibers was highest in the TAMs treated with AT-MSCs. Our finding of human-specific β-spectrin and dystrophin in hybrid myofibers containing human nuclei argues for myogenic programming of MSCs in regenerating murine skeletal muscle. For the further development of MSC-based treatments of myopathies, AT-MSCs appear to be the best choice in view of their efficient contribution to myoregeneration, their high ex vivo expansion potential, and because their harvesting is less demanding than that of BM- or SM-MSCs.