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.

Abnormal fibrillin assembly by dermal fibroblasts from two patients with Marfan syndrome.

The microfibrillar glycoprotein fibrillin is linked to the Marfan syndrome, an autosomal dominant connective tissue disorder. In this study, fibrillin synthesis, deposition and assembly has been investigated in Marfan dermal fibroblast lines from two unrelated patients for whom distinct mutations in the fibrillin gene FBN1 have been identified. In patient NB, a point mutation has occurred which causes an amino acid substitution and the other patient (GK) has a deletion in one allele. The two cell lines were broadly comparable with respect to de novo fibrillin synthesis and its distribution between medium and cell layer compartments. Electrophoresis of fibrillin immunoprecipitates confirmed the presence of fibrillin in medium and cell layers. GK cells secreted an additional higher relative molecular mass fibrillin-immunoreactive component. The time-course of fibrillin secretion was similar for the two lines, but differences in fibrillin aggregation were apparent. Rotary shadowing electron microscopy of extracted cell layers demonstrated the presence of abundant and extensive microfibrils in NB cell layers. These were abnormal in their gross morphology in comparison to microfibrils isolated from control cultures. No periodic microfibrillar structures were isolated from GK cell layers. These studies underline the need to classify fibrillin defects in terms of biochemical and ultrastructural criteria. Examination of the effects of individual mutations on microfibril organization will be particularly informative in elucidating the relationship between microfibril dysfunction and the complex clinical manifestations of Marfan patients.

Type VI collagen microfibrils: evidence for a structural association with hyaluronan.

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.

Calcium determines the supramolecular organization of fibrillin-rich microfibrils.

Microfibrils are ubiquitous fibrillin-rich polymers that are thought to provide long-range elasticity to extracellular matrices, including the zonular filaments of mammalian eyes. X-ray diffraction of hydrated bovine zonular filaments demonstrated meridional diffraction peaks indexing on a fundamental axial periodicity (D) of approximately 56 nm. A Ca2+-induced reversible change in the intensities of the meridional Bragg peaks indicated that supramolecular rearrangements occurred in response to altered concentrations of free Ca2+. In the presence of Ca2+, the dominant diffracting subspecies were microfibrils aligned in an axial 0.33-D stagger. The removal of Ca2+ caused an enhanced regularity in molecular spacing of individual microfibrils, and the contribution from microfibrils not involved in staggered arrays became more dominant. Scanning transmission electron microscopy of isolated microfibrils revealed that Ca2+ removal or addition caused significant, reversible changes in microfibril mass distribution and periodicity. These results were consistent with evidence from x-ray diffraction. Simulated meridional x-ray diffraction profiles and analyses of isolated Ca2+-containing, staggered microfibrillar arrays were used to interpret the effects of Ca2+. These observations highlight the importance of Ca2+ to microfibrils and microfibrillar arrays in vivo.

The Tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils.

Mice carrying the Tight skin (Tsk) mutation harbor a genomic duplication within the fibrillin-1 (Fbn 1) gene that results in a larger than normal in-frame Fbn 1 transcript. In this study, the consequences of the Tsk mutation for fibrillin-containing microfibrils have been examined. Dermal fibroblasts from Tsk/+ mice synthesized and secreted both normal fibrillin (approximately 330 kD) and the mutant oversized Tsk fibrillin-1 (approximately 450 kD) in comparable amounts, and Tsk fibrillin-1 was stably incorporated into cell layers. Immunohistochemical and ultrastructural analyses of normal and Tsk/+ mouse skin highlighted differences in the gross organization and distribution of microfibrillar arrays. Rotary shadowing of high Mr preparations from Tsk/+ skin demonstrated the presence of abundant beaded microfibrils. Some of these had normal morphology and periodicity, but others were distinguished by diffuse interbeads, longer periodicity, and tendency to aggregate. The presence of a structurally abnormal population of microfibrils in Tsk/+ skin was unequivocally demonstrated after calcium chelation and in denaturating conditions. Scanning transmission electron microscopy highlighted the presence of more mass in Tsk/+ skin microfibrils than in normal mice skin microfibrils. These data indicate that Tsk fibrillin-1 polymerizes and becomes incorporated into a discrete population of beaded microfibrils with altered molecular organization.

The supramolecular organization of fibrillin-rich microfibrils.

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

A mutation in FBN1 disrupts profibrillin processing and results in isolated skeletal features of the Marfan syndrome.

Dermal fibroblasts from a 13-yr-old boy with isolated skeletal features of the Marfan syndrome were used to study fibrillin synthesis and processing. Only one half of the secreted profibrillin was proteolytically processed to fibrillin outside the cell and deposited into the extracellular matrix. Electron microscopic examination of rotary shadowed microfibrils made by the proband's fibroblasts were indistinguishable from control cells. Sequencing of the FBN1 gene revealed a heterozygous C to T transition at nucleotide 8176 resulting in the substitution of a tryptophan for an arginine (R2726W), at a site immediately adjacent to a consensus sequence recognized by a cellular protease. Six other individuals in the proband's family had the FBN1 mutation that segregated with tall stature. None of the affected individuals have cardiac or ocular manifestations of the Marfan syndrome. This mutation identifies a putative site for profibrillin to fibrillin processing, and is associated with isolated skeletal features of the Marfan syndrome, indicating that the FBN1 gene is one of the genes that determines height in the general population. The cellular effect of the mutation may be equivalent to a "null" FBN1 allele and may define the phenotype associated with FBN1 "null" alleles.

The molecular genetics of Marfan syndrome and related disorders.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

Truncated profibrillin of a Marfan patient is of apparent similar size as fibrillin: intracellular retention leads to over-N-glycosylation.

We studied profibrillin-1 (proFib) synthesis and microfibril formation in cultured fibroblasts from an individual with severe Marfan syndrome harboring a premature stop codon (W2756ter) in one FBN1 allele. Rotary shadowing analysis of extracellular matrix produced by these cells revealed the presence of only a very few intact microfibrils which showed marked disorganisation within the interbeaded domains. Metabolic pulse-chase studies identified intracellularly a population of truncated proFib molecules which were secreted more slowly than the normal proFib derived from the normal allele. Culture media contained strikingly reduced amounts of wild-type proFib in comparison to fibrillin (Fib). Our findings imply that (1) the truncated proFib is secreted and disturbs microfibril assembly; (2) the mutation is probably close to a putative cleavage site in the proFib C terminus necessary for the conversion of proFib to Fib; (3) the truncated proFib is over-N-glycosylated due to intracellular retention rather than incomplete cleavage of proFib with persistence of N-glycosylated sites; (4) not all potential N-glycosylation sites in proFib seem to be normally used, since we could produce over-N-glycosylated proFib in normal cells by brefeldin A mediated intracellular captivation and subsequent appearance of over-glycosylated Fib in culture medium upon removal of the compound. It is conceivable that post-translational over-modification might be important for modulating the phenotype of FBN1 mutations in Marfan syndrome.

Fibrillin-rich microfibrils are reduced in photoaged skin. Distribution at the dermal-epidermal junction.

Chronic sun exposure results in photoaged skin with deep coarse wrinkles and loss of elasticity. We have examined the distribution and abundance of fibrillin-rich microfibrils, key structural components of the elastic fiber network, in photoaged and photoprotected skin. Punch biopsies taken from photoaged forearm and from photoprotected hip and upper inner arm of 16 subjects with a clinical range of photoaging were examined for fibrillin-1 and fibrillin-2 expression and microfibril distribution. In situ hybridization revealed decreased fibrillin-1 mRNA but unchanged fibrillin-2 mRNA levels in severely photoaged forearm biopsies relative to photoprotected dermal sites. An immunohistochemical approach demonstrated that microfibrils at the dermal-epidermal junction were significantly reduced in moderate to severely photoaged forearm skin. Confocal microscopy revealed that the papillary dermal microfibrillar network was truncated and depleted in photoaged skin. These studies highlight that the fibrillin-rich microfibrillar network associated with the upper dermis undergoes extensive remodeling following solar irradiation. These changes may contribute to the clinical features of photoaging, such as wrinkle formation and loss of elasticity.

A short-term screening protocol, using fibrillin-1 as a reporter molecule, for photoaging repair agents.

Photoaged skin is characterized by coarse and fine wrinkles. The mechanisms of wrinkle formation are undetermined, but appear to be due to changes within the matrix of the dermis and at the dermal-epidermal junction. Previous studies have identified marked reductions in procollagens I and III, collagen VII, and the fibrillin-rich microfibrillar apparatus in this area. Topically applied all-trans retinoic acid can repair photoaged dermal matrix, but this takes at least 6 mo of treatment. In this study, we have examined the abundance and distribution of fibrillin-1 prior to, and following, 192 wk of all-trans retinoic acid treatment. We have further developed a short-term protocol to determine the utility of potential repair agents, using fibrillin-1 as the marker for outcome. Individuals with clinically assessed severe photoaging were recruited to the study (n = 8). 0.025% all-trans retinoic acid, 5% sodium lauryl sulfate (irritant control), or vehicle were applied under occlusion to photoaged extensor forearm. A fourth control area was also occluded. After 96 h, punch biopsies were taken under local anesthesia and processed for either transmission electron microscopy or snap frozen. Frozen sections were prepared for immunohistochemistry and in situ hybridization immunohistochemistry. Electron microscopy revealed aberrant elastic fibers in the papillary dermis of photoaged forearm skin, with sparse microfibrillar apparatus and interstitial collagen. After application of 0.025% all-trans retinoic acid, there was increased deposition of both these dermal matrix components, with the aberrant elastic fibers no longer apparent. Significant increases (p < 0.05) were observed at the protein and mRNA levels for fibrillin-1 following all-trans retinoic acid and sodium lauryl sulfate treatments, with all-trans retinoic acid having a significantly greater effect than irritant control (p < 0.001); however, neither application had significant effect on the abundance of collagen VII or its mRNA. Investigation of collagen I synthesis revealed no difference following treatments. To ascertain the clinical relevance of using fibrillin-1 as a marker for photoaging, facial skin was biopsied at baseline and after long-term (192 wk) topical all-trans retinoic acid treatment (n = 5). Biopsies were wax-embedded and sections prepared for immunohistochemistry for fibrillin-1. Significant increases in the abundance of the microfibrillar apparatus was observed proximal to the dermal- epidermal junction (p < 0.001) following long-term all-trans retinoic acid application. This study indicates that all-trans retinoic acid can significantly affect fibrillin-1 content in photoaged skin. Furthermore, fibrillin-1 can be used as a "reporter" molecule in short-term protocols for testing the utility of topical agents in the repair of photoaged skin.

Fibrillin-containing microfibrils: structure and function in health and disease.

Fibrillin-containing microfibrils are a unique class of connective tissue macromolecules whose critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the recent linkage of their principal structural component fibrillin to Marfan syndrome, a heritable disorder with pleiotrophic connective tissue manifestations. The complexity of the structure: function relationships of these macromolecules was highlighted by the recent elucidation of the primary structure of fibrillin and characterisation of fibrillin mutations in Marfan patients. This review examines current understanding of the expression and assembly of fibrillin and describes new approaches which are now being applied to elucidate the many outstanding structural, organisational and functional aspects of the fibrillin-containing microfibrils.

Type VIII collagen: heterotrimeric chain association.

Two chains, alpha1(VIII) and alpha2(VIII), have been described for type VIII collagen. Early work suggested that these chains were present in a 2:1 ratio, although recent work has shown that homotrimers can form and predominate in some tissues. In order to address the question of whether the alpha1(VIII) and alpha2(VIII) chains could co-polymerise we made a shortened alpha1(VIII) chain and expressed this with full length alpha2(VIII) chain in an in vitro translation system supplemented with semi-permeabilised cells. Heterotrimers containing either two or one alpha2(VIII) were evident. Interestingly, a point mutation in the NC1 domain of the alpha1(VIII) chain abrogated trimer formation. In addition we were able to demonstrate chain association of the alpha1(X) chain of type X collagen with the shortened alpha1(VIII) chain. Variations in chain association were seen when altered ratios of message were used. These results demonstrate the importance of the NC1 domain in chain association and suggest that gene expression regulates the composition and function of type VIII collagen by varying chain composition.

Scanning transmission electron microscopy mass analysis of fibrillin-containing microfibrils from foetal elastic tissues.

We have applied scanning transmission electron microscopy to intact native fibrillin-containing microfibrils isolated from foetal bovine elastic tissues in order to derive new insights into microfibril organisation. This technique provides quantitative data on the mass per unit length and axial mass distribution of unstained, unshadowed macromolecules. Scanning transmission electron microscopy of microfibrils from aorta, skin and nuchal ligament revealed that the beads corresponded to peaks of mass and the interbead regions to troughs of mass. These major features of axial mass distribution were characteristic of all microfibrils examined. Tissue-specific and age-dependent variations in mass were identified in microfibrils that were structurally comparable by rotary shadowing electron microscopy. Increased microfibril mass correlated with increasing gestational age. The additional mass was associated predominantly at, or close to, the bead. Some microfibril populations exhibited pronounced assymetry in their axial mass distribution. These data indicate that intact native microfibrillar assemblies from developing elastic tissues are heterogeneous in composition. Loss of mass following chondroitinase ABC or AC lyase treatment confirmed the presence of chondroitin sulphate in nuchal ligament microfibrillar assemblies.

Cellular and extracellular biology of the latent transforming growth factor-beta binding proteins.

The latent transforming growth factor-beta binding proteins (LTBP) are a recently identified family of widely expressed multidomain glycoproteins that range in size from 125 kDa to 240 kDa. Four LTBP genes have been described, and the homology of latent transforming growth factor-beta binding proteins molecules to the fibrillins has resulted in their inclusion in the so-called 'fibrillin superfamily'. They form intracellular covalent complexes with latent transforming growth factor-beta and target these growth factors to the extracellular matrix. This review describes their structure, summarizes current understanding of their dual roles as growth factor binding proteins and components of the extracellular matrix, and highlights their significance in tissue development and disease.

Fibrillin secretion and microfibril assembly by Marfan dermal fibroblasts.

The Marfan syndrome has been linked to the FBN1 gene encoding the microfibrillar glycoprotein fibrillin. To date, there have been no descriptions of microfibrillar abnormalities characteristic of this connective tissue disorder, although biochemical analyses have highlighted apparent abnormalities in fibrillin synthesis, secretion and processing. We have conducted a biochemical and ultrastructural investigation of fibrillin expression and assembly by a panel of dermal fibroblast lines from patients with Marfan syndrome and related diseases. The study has highlighted marked differences between cells in terms of secretion and aggregation of newly-synthesised fibrillin. In addition, electron microscopic visualization of fibrillin assemblies has clearly demonstrated for the first time the plethora of microfibrillar abnormalities that underlie this heterogeneous disorder. These data emphasize the molecular complexity that is a feature of the diverse clinical phenotypes exhibited by Marfan patients.

The role of calcium in the organization of fibrillin microfibrils.

The microfibrillar glycoprotein fibrillin has a multidomain structure which contains forty-three epidermal growth factor-like motifs with calcium-binding consensus sequences. We have utilized intact microfibrils isolated from human dermal fibroblast cultures to investigate the putative influence of bound calcium on microfibrillar organization and integrity. Incubation with EDTA or EGTA rapidly resulted in gross disruption of microfibril morphology. The treatment induced disorganization of the interbead domains although the regular beaded arrangement was always apparent. These changes were readily reversible on replacing calcium, indicating that the treatment had not compromised microfibrillar integrity. The data localize calcium binding EGF-like repeats to the interbead domains and indicate that lateral packing of fibrillin monomers is calcium-dependent. This arrangement suggests how mutations in epidermal growth factor-like domains of fibrillin might cause the disruption in microfibril organization and interactions which underlies the clinical symptoms of some Marfan syndrome patients.

Fibrillin-rich microfibrils: an X-ray diffraction study of the fundamental axial periodicity.

Microfibrils are ubiquitous matrix polymers which are thought to provide elastic properties in all extracellular matrix structures. The major component of the elastic microfibrils is the protein fibrillin; its molecular structure is unknown. In electron microscopy, microfibrils appear as beaded structures exhibiting a variable periodicity, indicating that they may be elastomeric. The X-ray diffraction of fibrillin-rich microfibrils in the form of zonular filaments from bovine eyes exhibits meridional diffraction peaks indexing on a fundamental periodicity of 55 nm in the relaxed state. The application of a 40% extension produced a lengthening of the periodicity by 3% as judged by alteration of the D spacing of the principal peaks. This effect was shown to be reversible. Changes in the periodicity of the meridional reflections indicate changes in the fundamental structure of the microfilaments, but cannot account for all long range elastomeric properties of fibrillin-containing microfibrils.

Fibrillin: evidence that chondroitin sulphate proteoglycans are components of microfibrils and associate with newly synthesised monomers.

We have investigated the potential association of proteoglycans with intact fibrillin-containing microfibrils from foetal bovine elastic tissues and with newly synthesised fibrillin in human and bovine cell cultures. Microfibril integrity was disrupted by chondroitinase ABC lyase and chondroitinase AC lyase, but not by keratanase or hyaluronidase. Following chondroitinase treatment, beads were disrupted but the underlying fibrillar scaffold appeared intact. Cuprolinic blue was prominently associated with beaded domains at a critical electrolyte concentration. Electron-dense rods were often associated with cuprolinic blue-treated microfibrils isolated from fixed tissues. Positive staining revealed charged foci at the beads. Newly synthesised fibrillin could be labelled with 35S TransLabel, [3H]glucosamine or 35SO4 but its electrophoretic mobility was not influenced by treatment with chondroitinase ABC or AC lyase. A diffuse 35SO4-labelled chondroitinase-sensitive component with a resistant band (Mr 35000) co-immunoprecipitated with fibrillin. These experiments indicate that chondroitin sulphate proteoglycans associate with fibrillin and contribute to microfibril assembly. This association has major implications for microfibril function in health and disease.

Embryonic chick cartilage collagens. Differences in the low-Mr species present in sternal cartilage and tibiotarsal articular cartilage.

The collagenous polypeptides present in embryonic chick sternal and tibiotarsal cartilages have been solubilised by digestion with pepsin and separated by salt fractionation. Type II collagen, 1 alpha 2 alpha 3 alpha collagen, and two polypeptides (apparent molecular mass 150 and 42 kDa), which were reducible to a number of smaller peptides, were extracted from both tissues. However, also present in the peptic digests of tibiotarsal cartilages was a major non-reducible highly-soluble polypeptide of 45 kDa. This short-chain collagen is apparently identical to the pepsinized product of G collagen (Mr 59 000), a major low-Mr procollagen-like species previously detected in chick chondrocyte cultures.