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Spinal cord injury results in disruption of the cord microstructure, which is followed by inflammation leading to additional deterioration. Perivascular basement membranes are a component of the spinal cord microstructure that lies between blood vessels and astrocytes. The impact of disrupting the basement membrane structure on the expansion of inflammation has not been fully examined. The objective of this study was to clarify the relationship between damage to basement membranes and inflammation after spinal cord injury. Immunohistochemical analyses of the perivascular extracellular matrix were performed in a mouse spinal cord injury model. In normal tissue, the perivascular basement membrane was a single-layer structure produced by both endothelial cells and surrounding astrocytes. After spinal cord injury, however, the perivascular basement membrane often separated into an inner endothelial basement membrane and an outer parenchymal basement membrane. The altered basement membranes formed during the acute phase (within 7 days after spinal cord injury). During the subacute phase of injury, numerous monocytes and macrophages accumulated in the space between the separated basement membranes and infiltrated into the parenchyma where astrocytic endfeet were displaced. Infiltration of inflammatory cells from the injury core was attenuated coincident with the appearance of the glia limitans and glial scar. Furthermore, the outer parenchymal basement membrane was connected to the basement membrane of the glia limitans surrounding the injury core. Our data suggest that structurally altered basement membranes facilitate expansion of secondary inflammation during the subacute phase of spinal cord injury.

In the ischemic brain, reperfusion with tissue plasminogen activator (tPA) sometimes causes catastrophic hemorrhagic transformation (HT); however, the mechanism remains elusive. Here, we show that the basement membrane, and not the endothelial cells, is vulnerable to ischemic/reperfusion injury with tPA treatment. We treated a spontaneously hypertensive rat model of middle cerebral artery occlusion (MCAO) with vehicle alone, tPA alone, or a free radical scavenger, edaravone, plus tPA. Light and electron microscopic analyses of each microvascular component revealed that the basement membrane disintegrated and became detached from the astrocyte endfeet in tPA-treated animals that showed HT. On the other hand, edaravone prevented the dissociation of the neurovascular unit, dramatically decreased the HT, and improved the neurologic score and survival rate of the tPA-treated rats. These results suggest that the basement membrane that underlies the endothelial cells is a key structure for maintaining the integrity of the neurovascular unit, and a free-radical scavenger can be a viable agent for inhibiting tPA-induced HT.

In the present study, we have analyzed the alpha(IV) chain distribution in the subepithelial basement membrane (BM) of the rat pulmonary airway from the bronchi to alveoli. We have furthermore analyzed the alpha(IV) chain distribution in the subepithelial BM of the bronchioalveolar duct junction (BADJ) using alpha(IV) chain specific monoclonal antibodies. Our results show that the BM of the bronchial and bronchiolar epithelium contains [alpha1(IV)]2alpha2(IV) and [alpha5(IV)]2alpha6(IV) molecules and confirmed that the alveolar BM consists of [alpha1(IV)]2alpha2(IV) and alpha3(IV) alpha4(IV)alpha5(IV) molecules. There are also small regions in BADJ consisting of only [alpha1(IV)]2alpha2(IV) molecules without alpha3(IV)alpha4(IV)alpha5(IV) and [alpha5(IV)]2alpha6(IV) molecules. Moreover, the bronchioalveolar stem cells (BASCs)-primordial cells for bronchiolar Clara cells and alveolar type II (AT2) cells - lie adjacent to such small regions. These findings suggest that [alpha1(IV)]2 alpha2(IV) may be important for the BASCs to self-renew or to self-maintain themselves and that microenvironments produced by alpha(IV) chains may be important for cell differentiation.

We studied distribution patterns of type IV collagen alpha chains in the subepithelial basement membrane (SBM) of the human gastrointestinal tract - the esophagus through the anal canal - by immunofluorescent microscopy using alpha(IV) chain-specific monoclonal antibodies. The alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains were found in the SBM throughout the tract, indicating the localization of [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV) heterotrimeric molecules. The [alpha1(IV)](2)alpha2(IV) molecule was continuously stained, while the [alpha5(IV)](2)alpha6(IV) molecule was weakly stained in gastric glands and small intestinal crypts. In addition, the SBM at the luminal surface epithelium of the stomach and large intestine contained small amounts of alpha3(IV) and alpha4(IV) chains which combined to form the alpha3(IV)alpha4(IV)alpha5(IV) heterotrimeric molecule with alpha5(IV) chain. The SBM beneath the villous epithelium of the small intestine was also demonstrated to have an alpha3(IV) chain and alpha4(IV) chain. Considering the specific locations of the type IV collagen trimers throughout the gastrointestinal SBM, the supramolecular network containing the alpha3(IV)alpha4(IV)alpha5(IV) molecule appears to function as a selective permeability barrier and /or as a protection against chemical stress from the luminal digestive enzymes.

Distributions of type IV collagen alpha chains in the basement membrane (BM) of human skin and its appendages were analyzed by immunofluorescent microscopy using chain-specific monoclonal antibodies. The basement membrane beneath the epidermis contained [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV) but no alpha3(IV)alpha4(IV)alpha5(IV); this held true for at the eccrine sweat glands and glandular ducts, sebaceous glands, hair follicles, and arrector muscles of hair. The secretary portion of the eccrine sweat glands was rich in [alpha1(IV)](2) alpha2(IV) and had less [alpha5(IV)](2)alpha6(IV), while [alpha5(IV)](2) alpha6(IV) was abundant in the ductal portion. In the subepidermal zone, alpha5(IV)/alpha6(IV) chain negative spots (1.9-15.0 microm) were frequently observed. Triple staining samples (Mel.2, alpha2(IV) and alpha5(IV) chains) showed that about 50% of epidermal melanocytes colocalized with such spots. Results suggest that these alpha5(IV)/alpha6(IV) chain negative spots of the subepidermal basement membrane have a particular relationship with melanocytes and are sites for certain interactions between the two.

Abstract: Vertebrate collagen types XV and XVIII are broadly distributed basement membrane components, classified into a structurally distinct subgroup called “multiplexin collagens”. Mutations in mammalian multiplexins are identified in some degenerative diseases such as Knobloch syndrome 1 (KNO1) or skeletal/cardiac myopathies, however, these progressive properties have not been elucidated. Here we investigated Drosophila mutants of Multiplexin (Mp), the only orthologue of vertebrate collagen types XV and XVIII, to understand the pathogenesis of multiplexin-related diseases. The mp mutants exhibited morphological changes in cardiomyocytes and progressive dysfunction of the skeletal muscles, reminiscent phenotypes observed in Col15a1-null mice. Ultrastructural analysis revealed morphologically altered mitochondria in mutants’ indirect flight muscles (IFMs), resulting in severely attenuated ATP production and enhanced reactive oxygen species (ROS) production. In addition, mutants’ IFMs exhibited diminished βPS integrin clustering and abolished focal adhesion kinase (FAK) phosphorylation. Furthermore, mutants’ defective IFMs are improved by the administrations of cyclosporin A, an inhibitor against mitochondrial permeability transition pore (mPTP) opening or losartan, an angiotensin II type 1 receptor (AT1R) blocker. Thus, our results suggest that Mp modulates mPTP opening and AT1R activity through its binding to integrin and that lack of Mp causes unregulated mPTP opening and AT1R activity, leading to mitochondrial dysfunctions. Hence, our results provide new insights towards the roles of multiplexin collagens in mitochondrial homeostasis and may serve as pharmacological evidences for the potential use of cyclosporin A or losartan for the therapeutic strategies. [Copyright &y& Elsevier]

Abstract: Multiplexin (Mp) is the Drosophila orthologue of vertebrate collagens XV and XVIII. Like them, Mp is widely distributed in the basement membranes of the developing embryos, including those of neuroblasts in the central and peripheral nervous systems, visceral muscles of the gut, and contractile cardioblasts. Here we report the identification of mutant larvae bearing piggyBac transposon insertions that exhibit decrease Mp production associated with abdominal cuticular and wing margin defects, malformation of sensory organs and impaired sensitivity to physical stimuli. Additional findings include the abnormal ultrastructure of fatbody associated with abnormal collagen IV deposition, and reduced Wingless deposition. Collectively, these findings are consistent with the notion that Mp is required for the proper formation and/or maintenance of basement membrane, and that Mp may be involved in establishing the Wingless signaling gradients in the Drosophila embryo. [Copyright &y& Elsevier]