Image-based assessment of microvascular function and structure in collagen XV- and XVIII-deficient mice.

Collagen XV and XVIII are ubiquitous constituents of basement membranes. We aimed to study the physiological roles of these two components of the permeability barrier non-invasively in striated muscle in mice deficient in collagen XV or XVIII by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Structural information was obtained with transmission electron microscopy (TEM). MR data were analysed by two different analysis methods to quantify tissue perfusion and microcirculatory exchange parameters to rule out data analysis method-dependent results. Control mice (C57BL/6J Ola/Hsd strain) or mice lacking either collagen XV (Col15a1(-/-)) or XVIII (Col18a1(-/-)) were included in the study. MR images were acquired using a preclinical system using gadodiamide (Gd-DTPA-BMA, molecular weight 0.58 kDa) as a tracer. Exchange capacity (permeability (P)-surface area (S) product relative to blood flow (FB)) was increased in test mice compared to controls, but the contributions from P, S, and FB were different in these two phenotypes. FB was significantly increased in Col18a1(-/-), but slightly decreased in Col15a1(-/-). PS was significantly increased only in Col18a1(-/-) even though P was increased in both phenotypes suggesting S might also be reduced in Col15a1(-/-) mice. Immunohistochemistry and electron microscopy demonstrated alterations in capillary density and morphology in both knockout mouse strains in comparison to the control mice. Both collagen XV and XVIII are important for maintaining normal capillary permeability in the striated muscle. DCE-MRI and the perfusion analyses successfully determined microvascular haemodynamic parameters of genetically modified mice and gave results consistent with more invasive methods.

Soluble endostatin is a novel inhibitor of epithelial repair in idiopathic pulmonary fibrosis.

BACKGROUND AND AIM: Aberrant angiogenesis and defective epithelial repair are key features of idiopathic pulmonary fibrosis (IPF). Endostatin is an antiangiogenic peptide with known effects on endothelial cells. This study aimed to establish the levels of endostatin in the bronchoalveolar lavage fluid (BALF) in IPF and to investigate its actions on distal lung epithelial cells (DLEC) and primary type II cells. METHODS: 20 patients with IPF and 10 controls underwent BAL. Endostatin was measured by ELISA. BALF cytokines and matrix metalloproteinase (MMP)-3 were measured by Luminex array. Primary DLEC monolayers were wounded and treated with endostatin. Apoptosis and cell viability were assessed. RESULTS: Endostatin was elevated in the BALF and plasma of patients with IPF compared with normal controls. There was a negative correlation between endostatin, forced vital capacity and gas transfer. Endostatin correlated with a number of proinflammatory cytokines and MMP3. Physiological endostatin doses inhibited DLEC wound repair by 44% in an effect that was partially FasL and caspase dependent. Endostatin increased apoptosis rates by 8% and reduced their viability by 34%. Similar effects of endostatin were seen in primary type II cells in terms of inhibition of wound repair and proliferation. CONCLUSIONS: Elevated BALF endostatin levels correlated with a number of elevated cytokines, MMP3 and lung function in IPF. Endostatin is a novel inhibitor of DLEC wound repair, inducing apoptosis and reducing cell viability in a FasL and caspase dependent manner. Endostatin may play a role in aberrant epithelial repair in IPF.

The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles.

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

Tumor hepatocytes and basement membrane-Producing cells specifically express two different forms of the endostatin precursor, collagen XVIII, in human liver cancers.

Endostatin is an endogenous inhibitor of angiogenesis and tumor growth in mice, which may be generated by proteolytic cleavage of collagen XVIII. In normal tissues, 2 variants of the endostatin precursor, namely the SHORT and LONG forms, regulate tissue specificity. We analyzed 53 human liver biopsies (18 hepatocellular carcinomas, 16 metastases of colorectal cancer, 3 cholangiocarcinomas, and 16 controls) by RNA dot blots, double-labeling immunohistochemistry, and in situ hybridization, using common and variant-specific probes. Tumor hepatocytes expressed the LONG form, whereas cholangiocarcinoma cells expressed the SHORT form, which was deposited in tumor basement membranes. Metastatic colorectal carcinoma cells did not express collagen XVIII. In the stromal compartment of primary and metastatic cancers, myofibroblasts and vascular endothelial cells expressed the SHORT form. Both basement membrane components, collagen IV and the SHORT collagen XVIII form, were codistributed and their mRNA levels strongly correlated (R =.75, P <.001). In addition, freshly isolated human hepatocytes expressed the LONG form and culture-activated stellate cells the SHORT form. Moreover, the full-length LONG form is a plasma protein. Thus, the LONG form is a hepatocyte-specific variant, and the SHORT form is a major component of the tumor extracellular matrix in primary and metastatic liver cancers. In the clinical context, the global expression of the endogenous endostatin precursor, collagen XVIII, in liver cancer results from the combined expression profiles of tumor cells, stromal cells, and nontumor hepatocytes at the advancing edge of the tumor, particular to each type of cancer.

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C.

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.

The vaccinia virus 39-kDa protein forms a stable complex with the p4a/4a major core protein early in morphogenesis.

The vaccinia virus (VV) 39-kDa protein, the product of the A4L gene, is a highly antigenic protein of the viral core. Pulse-chase and immunoprecipitation experiments have shown that the 39-kDa protein interacts with p4a (encoded by the A10L gene), the precursor of the most abundant virion protein. This interaction is maintained with the processed 4a form that arises during virion maturation. The controlled disruption of mature viral particles showed that the 39-kDa and 4a proteins are tightly bound within the virion. Immunoelectron microscopy showed that both proteins first localize within the cytoplasm and later accumulate inside the viral factories, reaching these locations via a mechanism apparently unrelated to cellular membranes. Double labeling experiments showed a colocalization of both proteins in all virus-induced structures.

Transgenic mice overexpressing ornithine and S-adenosylmethionine decarboxylases maintain a physiological polyamine homoeostasis in their tissues.

Recent work has shown that transgenic mice overexpressing human ornithine decarboxylase display no marked changes in the tissue concentrations of spermidine or spermine in spite of a dramatic increase in putrescine levels. In the tissues of transgenic mice carrying the human spermidine synthase gene and in those of hybrid mice overexpressing both ornithine decarboxylase and spermidine synthase, spermidine and spermine levels remain within normal limits. To test whether the amount of the propylamine group donor, decarboxylated S-adenosylmethionine, limits the conversion of putrescine into the higher polyamines, we have produced transgenic mouse lines harbouring the rat S-adenosylmethionine decarboxylase gene in their genome. However, neither these mice nor the hybrid mice overexpressing both ornithine decarboxylase and S-adenosylmethionine decarboxylase displayed significant changes in their spermidine and spermine tissue levels. To study the mechanism by which cells maintain the constancy of the polyamine concentrations, we have determined the metabolic flux of polyamines in transgenic primary fibroblasts using pulse labelling. The results indicate that the polyamine flow is faster in transgenic primary fibroblasts than in non-transgenic fibroblasts and that the intracellular homoeostasis of higher polyamines is maintained at least partly by the acetylation of spermidine and spermine and their secretion into the medium.