Mesenchymal stromal cells:inhibiting PDGF receptors or depleting fibronectin induces mesodermal progenitors with endothelial potential

Realizing the full therapeutic potential of mesenchymal stromal/stem cells (MSCs) awaits improved understanding of mechanisms controlling their fate. Using MSCs cultured as spheroids to recapitulate a three-dimensional cellular environment, we show that perturbing the mesenchymal regulators, platelet-derived growth factor (PDGF) receptors or fibronectin, reverts MSCs toward mesodermal progenitors with endothelial potential that can potently induce neovascularization in vivo. MSCs within untreated spheroids retain their mesenchymal spindle shape with abundant smooth muscle α-actin filaments and fibronectin-rich matrix. Inhibiting PDGF receptors or depleting fibronectin induces rounding and depletes smooth muscle α-actin expression; these cells have characteristics of mesenchymoangioblasts, with enhanced expression of mesendoderm and endoderm transcription factors, prominent upregulation of E-cadherin, and Janus kinase signaling-dependent expression of Oct4A and Nanog. PDGF receptor-inhibited spheroids also upregulate endothelial markers platelet endothelial cell adhesion molecule 1 and vascular endothelial-cadherin and secrete many angiogenic factors, and in vivo they potently stimulate neovascularization, and their MSCs integrate within functional blood vessels that are perfused by the circulation. Thus, MSC potency and vascular induction are regulated by perturbing mesenchymal fate

Fibrillin-1 regulates the bioavailability of TGFβ1

We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) β1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFβ signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a fibrillin-1 sequence encoded by exons 44–49 releases endogenous TGFβ1, thereby stimulating TGFβ receptor–mediated Smad2 signaling. This altered TGFβ1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFβ1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFβ1-releasing sequence specifically associates with full-length fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal fibrillin-1, thereby inhibiting the association of C-terminal latent TGFβ-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal fibrillin-1. By releasing LLC from microfibrils, the fibrillin-1 sequence encoded by exons 44–49 can contribute to MFS and related diseases

Type II and VI collagen in nasal and articular cartilage and the effect of IL-1α on the distribution of these collagens

The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1α (IL-1α). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen. Also in sections of explants cultured with IL-1α a higher level of labeling of the collagens was found. This was apparent without any pre-treatment with hyaluronidase. Under the influence of IL-1α the area positive for type VI collagen that surrounded the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion of this type of cartilage hardly occurred in the presence of IL-1α whereas nasal cartilage was almost completely degraded within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion

Identification of Mechanosensitive Genes during Embryonic Bone Formation

Although it is known that mechanical forces are needed for normal bone development, the current understanding of how biophysical stimuli are interpreted by and integrated with genetic regulatory mechanisms is limited. Mechanical forces are thought to be mediated in cells by “mechanosensitive” genes, but it is a challenge to demonstrate that the genetic regulation of the biological system is dependant on particular mechanical forces in vivo. We propose a new means of selecting candidate mechanosensitive genes by comparing in vivo gene expression patterns with patterns of biophysical stimuli, computed using finite element analysis. In this study, finite element analyses of the avian embryonic limb were performed using anatomically realistic rudiment and muscle morphologies, and patterns of biophysical stimuli were compared with the expression patterns of four candidate mechanosensitive genes integral to bone development. The expression patterns of two genes, Collagen X (ColX) and Indian hedgehog (Ihh), were shown to colocalise with biophysical stimuli induced by embryonic muscle contractions, identifying them as potentially being involved in the mechanoregulation of bone formation. An altered mechanical environment was induced in the embryonic chick, where a neuromuscular blocking agent was administered in ovo to modify skeletal muscle contractions. Finite element analyses predicted dramatic changes in levels and patterns of biophysical stimuli, and a number of immobilised specimens exhibited differences in ColX and Ihh expression. The results obtained indicate that computationally derived patterns of biophysical stimuli can be used to inform a directed search for genes that may play a mechanoregulatory role in particular in vivo events or processes. Furthermore, the experimental data demonstrate that ColX and Ihh are involved in mechanoregulatory pathways and may be key mediators in translating information from the mechanical environment to the molecular regulation of bone formation in the embryo

Assignment of the gene for cytosolic alanine aminotransferase (AAT1) to human chromosome 8

The segregation of human cytosolic alanine aminotransferase (AAT1) and the individual human chromosomes has been studied in 27 secondary and tertiary rat hepatoma-human (liver) fibroblast hybrids. The staining solution used to visualize AAT activity on starch gels was specific for AAT since it was visualized only when all components of the stain were present. The locus for human AAT1 has been assigned to chromosome 8

The Evolution of Extracellular Fibrillins and Their Functional Domains

Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and non-elastic extracellular matrices, and are known to interact with several binding partners including tropoelastin and integrins. Here, we study the evolution of fibrillin proteins. Following sequence collection from 39 organisms representative of the major evolutionary groups, molecular evolutionary genetics and phylogeny inference software were used to generate a series of evolutionary trees using distance-based and maximum likelihood methods. The resulting trees support the concept of gene duplication as a means of generating the three vertebrate fibrillins. Beginning with a single fibrillin sequence found in invertebrates and jawless fish, a gene duplication event, which coincides with the appearance of elastin, led to the creation of two genes. One of the genes significantly evolved to become the gene for present-day fibrillin-1, while the other underwent evolutionary changes, including a second duplication, to produce present-day fibrillin-2 and fibrillin-3. Detailed analysis of several sequences and domains within the fibrillins reveals distinct similarities and differences across various species. The RGD integrin-binding site in TB4 of all fibrillins is conserved in cephalochordates and vertebrates, while the integrin-binding site within cbEGF18 of fibrillin-3 is a recent evolutionary change. The proline-rich domain in fibrillin-1, glycine-rich domain in fibrillin-2 and proline-/glycine-rich domain in fibrillin-3 are found in all analyzed tetrapod species, whereas it is completely replaced with an EGF-like domain in cnidarians, arthropods, molluscs and urochordates. All collected sequences contain the first 9-cysteine hybrid domain, and the second 8-cysteine hybrid domain with exception of arthropods containing an atypical 10-cysteine hybrid domain 2. Furin cleavage sites within the N- and C-terminal unique domains were found for all analyzed fibrillin sequences, indicating an essential role for processing of the fibrillin pro-proteins. The four cysteines in the unique N-terminus and the two cysteines in the unique C-terminus are also highly conserved

Density of human bone marrow stromal cells regulates commitment to vascular lineages

Mechanisms underlying the vascular differentiation of human bone marrow stromal cells (HBMSCs) and their contribution to neovascularisation are poorly understood. We report the essential role of cell density-induced signals in directing HBMSCs along endothelial or smooth muscle lineages. Plating HBMSCs at high density rapidly induced Notch signaling, which initiated HBMSC commitment to a vascular progenitor cell population expressing markers for both vascular lineages. Notch also induced VEGF-A, which inhibited vascular smooth muscle commitment while consolidating differentiation to endothelial cells with cobblestone morphology and characteristic endothelial markers and functions. These mechanisms can be exploited therapeutically to regulate HBMSCs during neovascularisation