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

摘要

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.Key points class="unordered" style="list-style-type:disc">Collagen XV and XVIII occur in muscle and connective tissue capillaries and are needed for maintaining a normal circulatory phenotype.Lack of collagen XV in mice leads to increased vascular permeability, increased extraction fraction, and increased extravascular extracellular space in striated muscle.Lack of collagen XVIII in mice leads to increased blood flow, permeability, permeability–surface area product and blood–tissue transvascular transfer in striated muscle tissue.Our results show that functional imaging with MRI and subsequent data analysis provide reliable and robust data and are valuable tools for assessing detailed physiological information non-invasively. class="head no_bottom_margin" id="__sec2title">IntroductionIn recent years, our understanding of the vascular basement membranes (BMs) has changed dramatically, from being mere structural components of tissues and barriers to infiltration to acting as an active modulator of blood vessel formation and function, including the properties of the microcirculatory exchange barrier (Sund et al. ). The vascular BM of normal blood vessels is a uniform, well-defined sheet-like extracellular matrix underlying the endothelial cell layer and separating the endothelial cells from the stroma. It serves as an anchor for the pericytes and provides the vessels additional structural support (Kalluri, ; Sund et al. ). The BM surrounding the blood vessels also contributes to sequestering growth factors and angiogenic factors which influence the vasculature. The molecular composition characteristic of vascular BMs differs to some extent from the epithelial BMs (Kalluri, ) and there are also differences between the BMs of different vessels (Myllyharju & Kivirikko, ). The major components of the vascular BM include collagen IV, laminin, and proteoglycans which assemble to form a tight network that serves as a scaffold for endothelial cells (Kalluri, ; Sund et al. ). Collagen XV and XVIII are common constituents of most endothelial and epithelial BMs. The continuous capillaries, found in all muscular and connective tissues, have BMs containing both of these collagens (Tomono et al. ). Collagen XVIII has structural properties typical of collagens, which includes several non-collagenous domains, and also a proteoglycan with attached heparan sulphate side-chains. The closely related collagen XV is also glycosylated, with chondroitin-sulphate side-chains attached to a collagenous core protein (Iozzo, ). Together, collagen XV and XVIII form a distinct multiplexin group within the collagen superfamily (Myllyharju & Kivirikko, ) and their proteolytically cleaved C-terminal parts, named restin and endostatin are classified as endogenous angiogenesis inhibitors (Sund et al. ; Iozzo, ).Although these two types of collagen are structurally closely related, they appear to be functionally independent, with collagen XV prevalent in skeletal muscle and heart and collagen XVIII in the eye. Studies with knockout mice have shown that collagen XV is necessary for the structure and proper function of microvessels in the skeletal muscle, heart and in the skin (Eklund et al. ; Tomono et al. ; Rasi et al. ). On the other hand, mice lacking collagen XVIII have eye abnormalities and defects in the vasculature of the eye (Fukai et al. ; Marneros & Olsen, href="#b17" rid="b17" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_425073133">2003). In addition, abnormally broadened BMs have been observed in kidney proximal tubules, heart valves, epidermis and choroid plexus of collagen XVIII-deficient mice (Utriainen et al. href="#b31" rid="b31" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_425073147">1999). These observations indicate that this collagen is important for the structural integrity and function of endothelial and epithelial BMs (Utriainen et al. href="#b31" rid="b31" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_425073164">1999).Our hypothesis was that the circulatory phenotypes are dependant on the presence of collagen XV and/or collagen XVIII. We estimated blood volume, permeability, permeability–surface area products, extraction fraction (fraction of tracer removed from plasma in one vascular passage) and extracellular extravascular volume in knockout mice lacking either collagen XV (Col15a1^−/− mice) or collagen XVIII (Col18a1^−/− mice) using dynamic contrast-enhanced MRI (DCE-MRI) and subsequent perfusion analysis. Two different pharmacokinetic methods for analysis of perfusion properties were used in order to eliminate methods-dependent results. MR imaging methods may provide important information on the functional properties of the microcirculation both in healthy and diseased organs, are non-invasive so repeated studies of the same animal can be performed, and have a great translational value. We have recently reported a method (step–slope model) to determine the permeability of the capillary wall of a high molecular weight tracer using the initial tracer uptake in skin and skeletal muscle of mice lacking the atrial natriuretic peptide (ANP) receptor on endothelial cells (Curry et al. href="#b8" rid="b8" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_425073129">1984). The other perfusion analysis method using single-channel blind deconvolution with the adiabatic approximation model of Johnson and Wilson has been used to quantify capillary permeability, extraction fraction, extracellular extravascular volume and redistribution of the tracer back to the vasculature, as well as blood flow, blood volume and intravascular transit time in a region of interest (Taxt et al. href="#b27" rid="b27" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_425073132">2004). The present investigations are the first detailed application of this blind deconvolution approach in mouse models of altered microvascular function and are a step towards fulfilling the need for new non-invasive ways to study the regulation of microvascular exchange in whole animal tissues.Our new findings demonstrate that the striated muscle in mice lacking collagen XV or mice lacking collagen XVIII have increased blood-to-tissue leakage of a low molecular weight tracer although the relative contributions of increased perfusion and increased vascular permeability to the increased clearance differ between the two phenotypes. Light and electron microscopic analyses revealed abnormalities in the capillary density and ultrastructure of the capillaries in the knockout masseter muscle, which at least partially can explain the observed functional differences detected by MRI.