Absence of LTBP-3 attenuates the aneurysmal phenotype but not spinal effects on the aorta in Marfan syndrome.

Fibrillin-1 is an elastin-associated glycoprotein that contributes to the long-term fatigue resistance of elastic fibers as well as to the bioavailability of transforming growth factor-beta (TGFß) in arteries. Altered TGFß bioavailability and/or signaling have been implicated in aneurysm development in Marfan syndrome (MFS), a multi-system condition resulting from mutations to the gene that encodes fibrillin-1. We recently showed that the absence of the latent transforming growth factor-beta binding protein-3 (LTBP-3) in fibrillin-1-deficient mice attenuates the fragmentation of elastic fibers and focal dilatations that are characteristic of aortic root aneurysms in MFS mice, at least to 12 weeks of age. Here, we show further that the absence of LTBP-3 in this MFS mouse model improves the circumferential mechanical properties of the thoracic aorta, which appears to be fundamental in preventing or significantly delaying aneurysm development. Yet, a spinal deformity either remains or is exacerbated in the absence of LTBP-3 and seems to adversely affect the axial mechanical properties of the thoracic aorta, thus decreasing overall vascular function despite the absence of aneurysmal dilatation. Importantly, because of the smaller size of mice lacking LTBP-3, allometric scaling facilitates proper interpretation of aortic dimensions and thus the clinical phenotype. While this study demonstrates that LTBP-3/TGFß directly affects the biomechanical function of the thoracic aorta, it highlights that spinal deformities in MFS might indirectly and adversely affect the overall aortic phenotype. There is a need, therefore, to consider together the vascular and skeletal effects in this syndromic disease.

Comparison of 10 murine models reveals a distinct biomechanical phenotype in thoracic aortic aneurysms.

Thoracic aortic aneurysms are life-threatening lesions that afflict young and old individuals alike. They frequently associate with genetic mutations and are characterized by reduced elastic fibre integrity, dysfunctional smooth muscle cells, improperly remodelled collagen and pooled mucoid material. There is a pressing need to understand better the compromised structural integrity of the aorta that results from these genetic mutations and renders the wall vulnerable to dilatation, dissection or rupture. In this paper, we compare the biaxial mechanical properties of the ascending aorta from 10 murine models: wild-type controls, acute elastase-treated, and eight models with genetic mutations affecting extracellular matrix proteins, transmembrane receptors, cytoskeletal proteins, or intracellular signalling molecules. Collectively, our data for these diverse mouse models suggest that reduced mechanical functionality, as indicated by a decreased elastic energy storage capability or reduced distensibility, does not predispose to aneurysms. Rather, despite normal or lower than normal circumferential and axial wall stresses, it appears that intramural cells in the ascending aorta of mice prone to aneurysms are unable to maintain or restore the intrinsic circumferential material stiffness, which may render the wall biomechanically vulnerable to continued dilatation and possible rupture. This finding is consistent with an underlying dysfunctional mechanosensing or mechanoregulation of the extracellular matrix, which normally endows the wall with both appropriate compliance and sufficient strength.

Differential ascending and descending aortic mechanics parallel aneurysmal propensity in a mouse model of Marfan syndrome.

Marfan syndrome (MFS) is a multi-system connective tissue disorder that results from mutations to the gene that codes the elastin-associated glycoprotein fibrillin-1. Although elastic fibers are compromised throughout the arterial tree, the most severe phenotype manifests in the ascending aorta. By comparing biaxial mechanics of the ascending and descending thoracic aorta in a mouse model of MFS, we show that aneurysmal propensity correlates well with both a marked increase in circumferential material stiffness and an increase in intramural shear stress despite a near maintenance of circumferential stress. This finding is corroborated via a comparison of the present results with previously reported findings for both the carotid artery from the same mouse model of MFS and for the thoracic aorta from another model of elastin-associated glycoprotein deficiency that does not predispose to thoracic aortic aneurysms. We submit that the unique biaxial loading of the ascending thoracic aorta conspires with fibrillin-1 deficiency to render this aortic segment vulnerable to aneurysm and rupture.

Extracellular microfibrils in development and disease.

Fibrillins are the structural components of extracellular microfibrils that impart physical properties to tissues, alone or together with elastin as elastic fibers. Genetic studies in mice have revealed that fibrillin-rich microfibrils are also involved in regulating developmental programs and homeostatic processes through the modulation of TGF-beta/BMP signaling events. A new paradigm has thus emerged whereby the spatiotemporal organization of microfibrils dictates both the cellular activities and physical properties of connective tissues. These observations have paved the way to novel therapeutic approaches aimed at counteracting the life-threatening complications in human conditions caused by dysfunctions of fibrillin-rich microfibrils.

Osteopetrosis-like phenotype in latent TGF-beta binding protein 3 deficient mice.

LTBPs are extracellular matrix proteins resembling fibrillins. LTBP-1, 3, and 4 covalently bind latent TGF-beta and modulate tissue levels of this potent cytokine through regulation of its secretion, localization, and/or activation. To address LTBP function in vivo, we generated Ltbp-3 null mice. Ltbp-3-/- animals developed craniofacial abnormalities due to early ossification of the skull base synchondroses and displayed reduced body size. In addition, histological examination of Ltbp-3-/- skeletons revealed an increase in bone mass. The osteoblast numbers and mineral apposition rates were decreased in Ltbp-3-/- mice, whereas the osteoclast numbers were similar in null and wild type mice. Histological examination revealed persistence of cartilage remnants in Ltbp-3-/- trabecular bone. Taken together, these results indicate that the Ltbp-3-/- high bone mass phenotype was due to a defect in bone resorption. We hypothesize that lack of Ltbp-3 results in decreased levels of TGF-beta in bone and cartilage, which leads to compromised osteoclast function and decreased bone turnover.

Latent TGF-beta binding proteins.

Latent TGF-beta binding proteins are multidomain proteins with a common, highly repetitive structural organization and partially overlapping expression patterns. Latent TGF-beta binding protein-1, -3 and -4 bind latent TGF-beta. TGF-betas are normally secreted as latent complexes, consisting of the mature TGF-beta dimer non-covalently bound to its processed propeptide dimer plus a latent TGF-beta binding protein. The latent TGF-beta binding protein is covalently bound to the propeptide. These binding proteins may perform at least two functions: structural, as components of the matrix, and regulatory, as modulators of TGF-beta availability.

Bone defects in latent TGF-beta binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-beta presentation.

The latent transforming growth factor (TGF)-beta binding proteins (LTBP)-1, -3 and -4 bind the latent form of the multipotent cytokine TGF-beta. To examine the function of the LTBPs, we made a null mutation of Ltbp-3 by gene targeting. The homozygous mutant animals developed cranio-facial malformations by 12 days. By three months, there was a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. The mutant animals developed osteosclerosis of the long bones and vertebrae as well as osteoarthritis between 6 and 9 months of age. These latter phenotypic changes were similar to those described for mice that have impaired TGF-beta signaling. Thus, we suggest that Ltbp-3 plays an important role in regulating TGF-beta bioavailability as the phenotype of the Ltbp-3 null mouse appears to result from decreased TGF-beta signaling. Histological examination of the skulls from null animals revealed no effects on calvarial suture closure. However, the synchondroses in the skull base were obliterated within 2 weeks of birth. This is in contrast to the wild-type synchondroses, which remain unossified throughout the life of the animal and enable growth of the skull base through endochondral ossification. Histological changes in mutant basooccipital-basosphenoid synchondrosis were observed 1.5 days after birth. Compared with wild-type or heterozygous littermates, the basooccipital-basosphenoid synchondrosis of Ltbp-3 null mice contained increased numbers of hypertrophic chondrocytes. The expression of bone sialoprotein-1 (a marker for osteoblasts) was observed in cells surrounding the synchondrosis at postnatal day 1.5 indicating ectopic ossification. The expression of Indian hedgehog (Ihh) (a marker for chondrocytes committed to hypertrophic differentiation) was found through the basooccipital-basosphenoid synchondrosis, whereas the expression of parathyroid hormone related protein (PTHrP), which inhibits chondrocyte differentiation, appeared to be diminished in Ltbp-3 null mice. This suggests that Ltbp-3 may control chondrocyte differentiation by regulating TGF-beta availability. TGF-beta may regulate PTHrP expression either downstream of Ihh or independently of Ihh signaling.

Tamoxifen and estrogen effects on TGF-beta formation: role of thrombospondin-1, alphavbeta3, and integrin-associated protein.

We have found that the enhanced activation of latent TGF-beta by human breast carcinoma cell lines either treated with tamoxifen or deprived of estrogen is dependent upon thrombospondin-1 (TSP-1) since activation was blocked by anti-TSP-1 antibodies or by a TSP antagonist peptide. However, TGF-beta formation upon tamoxifen exposure to estrogen withdrawal is associated with decreased levels of soluble TSP-1. A concomitant increase in the expression of the TSP-1 receptors alphavbeta3 and integrin-associated protein (IAP) occurs under these conditions, and antibodies to TSP-1 or to these receptors inhibit increased TGF-beta formation. Therefore, increased cell surface associated TSP-1 enhances latent TGF-beta activation.

The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium.

The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism.

Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells.

Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides. Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc. On the basis of these results and on the structure of an eight-cysteine domain from fibrillin-1, we present a model of glycosylated CR3 and discuss the role of glycosylation in eight-cysteine domain protein-protein interactions.

Biochemical analysis of the arginine methylation of high molecular weight fibroblast growth factor-2.

The post-translational methylation of the N-terminally extended or high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) has been shown to affect the nuclear accumulation of the growth factor. In this study, we determined the extent and position of methyl groups in HMW FGF-2. Using mass spectrometry and amino acid sequence analysis, we have shown that the 22- and 22.5-kDa forms of HMW FGF-2 contain five dimethylated arginines located at positions -22, -24, -26, -36, and -38 using the methionine residue normally used to initiate the 18-kDa form as position 0. The 24-kDa form of HMW FGF-2 contains seven to eight dimethylated arginines located at positions -48, -50, and -52, in addition to positions -22, -24, -26, -36, and -38. In vitro methylation reactions demonstrate that the N-terminal extension of HMW FGF-2 acts as a specific substrate for yeast Hmt1p and human HRMT1L2 arginine methyltransferases. These findings indicate that HMW FGF-2, with the presence of five or more dimethylated Gly-Arg-Gly repeats, contains an RGG box-like domain, which may be important for protein-protein and/or protein-RNA interactions.

Modulation of cell growth and transformation by doxycycline-regulated FGF-2 expression in NIH-3T3 cells.

The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.

Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression.

The glycosylphosphatidylinositol (GPI)-anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts.

[Activation of latent TGF-beta. A required mechanism for vascular integrity]. / L'activation du TGF-beta latent. Un mécanisme requis pour l'intégrité vasculaire.

Recent molecular genetics studies in humans and mice showed that transforming growth factor-beta (TGF-beta) is involved in vasculogenesis and maintenance of blood vessel integrity. These results confirm earlier in vitro studies demonstrating generation of active TGF-beta when endothelial cells are cocultured with smooth muscle cells or pericytes. TGF-beta is secreted as a latent, inactive complex and becomes active only when released. Latent TGF-beta binds covalently to proteins (LTBP) that target it to the extracellular matrix. Thus, the latency of TGF-beta is essential to the regulation of the bioavailability and activity of this cytokine. The development of methods for measuring activation of latent TGF-beta in cell cultures and identification of the proteins contained in the latent TGF-beta complex have shed new light on the mechanism of activation of latent TGF-beta possibly involved in vasculogenesis, angiogenesis, and other processes.

Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals.

Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways.

Proteolytic control of growth factor availability.

Most growth factors are released from cells in a form that does not permit immediate interaction with their high affinity receptors. An important mechanism for presentation of these released latent growth factors is activation by the plasminogen activator-plasmin system. The involvement of this system in the biology of Transforming Growth Factor-beta (TGF-beta) is reviewed.

Diltiazem reduces retinal neovascularization in a mouse model of oxygen induced retinopathy.

PURPOSE: To assess the effect of diltiazem, a calcium channel blocking agent, on oxygen induced retinopathy (OIR) in a mouse model using neovascular nuclei quantitation and a quantitative scoring system based on examining fluorescein perfused retinal whole mount preparations. METHODS: The mouse model of oxygen induced retinopathy consisting of a 5 day exposure to 75% oxygen from postnatal day 7 to 12 was used to produce retinal neovascularization. Fluorescein conjugated dextran angiography of retinal vasculature was performed and retinal whole mounts were prepared to score features of retinopathy. The parameters that were scored in a masked fashion included blood vessel growth, blood vessel tuft formation, extra retinal neovascularization, degree of central vasoconstriction, retinal hemorrhage, and tortuosity of vessels. Diltiazem (0.05-0.5 mg/kg/day subcutaneously for five days) was administered to mice pups during exposure to oxygen to determine if calcium channel blockade altered retinopathy. In addition, quantification of retinal neovascular nuclei was performed in a masked fashion with periodic acid Schiff (PAS) staining of frozen eye sections. RESULTS: Animals that were exposed to hyperoxia for five days had a median (25th, 75th quartile) retinopathy score of 9 (8,11) versus control animals that had a retinopathy score of 1 (0,1) with p<0.001. Subscores for blood vessel growth, blood vessel tufts, extra-retinal neovascularization, central vasoconstriction, hemorrhage, and blood vessel tortuosity were all significantly different between control and treated animals. In addition, quantification of neovascular nuclei showed a significant increase in the number of nuclei extending beyond the inner limiting membrane into the vitreous in hyperoxic treated animals. Diltiazem at doses of 0.2 and 0.5 mg/kg/day improved the retinopathy as measured by the total retinopathy score [5 (4,6) and 4 (3.75,5.25), respectively]. The average number of extraretinal neovascular nuclei per retinal section (mean +/-standard deviation) was significantly decreased by diltiazem at doses of 0.2 and 0.5 mg/kg/day (31.4+/-18.8 and 20.9+/-6.9, respectively) when compared to hyperoxic treated animals (56.1+/-21.5). CONCLUSIONS: Diltiazem reduces oxygen induced retinopathy in the mouse as measured by a scoring system based on a retinal whole mount method of retinal neovascularization and by quantification of extra retinal neovascular nuclei.

The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis.

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.