Vascular Collagen Type-IV in Hypertension and Cerebral Small Vessel Disease

Background: Cerebral small vessel disease (SVD) is common in older people and causes lacunar stroke and vascular cognitive impairment. Risk factors include old age, hypertension and variants in the genes COL4A1/COL4A2 encoding collagen alpha-1(IV) and alpha-2(IV), here termed collagen-IV, which are core components of the basement membrane. We tested the hypothesis that increased vascular collagen-IV associates with clinical hypertension and with SVD in older persons and with chronic hypertension in young and aged primates and genetically hypertensive rats. Methods: We quantified vascular collagen-IV immunolabeling in small arteries in a cohort of older persons with minimal Alzheimer pathology (N=52; 21F/31M, age 82.8±6.95 years). We also studied archive tissue from young (age range 6.2–8.3 years) and older (17.0–22.7 years) primates (M mulatta) and compared chronically hypertensive animals (18 months aortic stenosis) with normotensives. We also compared genetically hypertensive and normotensive rats (aged 10–12 months). Results: Collagen-IV immunolabeling in cerebral small arteries of older persons was negatively associated with radiological SVD severity (ρ: −0.427, P=0.005) but was not related to history of hypertension. General linear models confirmed the negative association of lower collagen-IV with radiological SVD (P<0.017), including age as a covariate and either clinical hypertension (P<0.030) or neuropathological SVD diagnosis (P<0.022) as fixed factors. Reduced vascular collagen-IV was accompanied by accumulation of fibrillar collagens (types I and III) as indicated by immunogold electron microscopy. In young and aged primates, brain collagen-IV was elevated in older normotensive relative to young normotensive animals (P=0.029) but was not associated with hypertension. Genetically hypertensive rats did not differ from normotensive rats in terms of arterial collagen-IV. Conclusions: Our cross-species data provide novel insight into sporadic SVD pathogenesis, supporting insufficient (rather than excessive) arterial collagen-IV in SVD, accompanied by matrix remodeling with elevated fibrillar collagen deposition. They also indicate that hypertension, a major risk factor for SVD, does not act by causing accumulation of brain vascular collagen-IV.


Human cohort
Human tissue samples were supplied by Oxford Brain Bank (REC approval#15/SC/0639). Ethical approval for this study was provided by NHS-Research Ethics Service (Ref#12/EM/0028). We selected all cases from the Oxford Brain Collection with minimal Alzheimer's disease neuropathology (Braak stage 0-II) for whom brain imaging data were available.

Human clinical measurements
Medical history, including a documented history of hypertension, the use of antihypertensive medication (never/former/current) were collected from subjects and checked with family doctors' computerized records. History of hypertension was defined as systolic blood pressure >140 mmHg, or diastolic blood pressure >90 mmHg, or use of anti-hypertensive medication.

Radiological assessment of white matter lesions
Brain CT scans were performed on Siemens DR or ART scanners (Siemens AG, Munich, Germany). Scans were performed 591 [250, 1521] days prior to death (median [IQR]; range: 51 -4097 days). CT data for these cases have been previously reported 38,39 . In the present study, scans were reviewed at window width 80/40 for supratentorial slices (10mm slices). The severity of white matter lesions was rated on a categorical scale, range: 0/1/2/3, based on previously published scales 21,22 . A score of 0 indicated absence of hypodensity; 1 indicated questionable hypodensity, considered normal for age; 2 indicated periventricular hypodensity; 3 indicated marked hypodensity reaching the cortical grey matter. Scans were independently rated by an experienced neuroradiologist (Dr Rita Marasco) and by a neurologist with expertise in cerebrovascular disease (G.Z.). In the event of discordant ratings, cases were reviewed and consensus reached in all cases.
Neuropathological assessment of SVD Assignment to neuropathological "SVD" was based on microscopic examination of haematoxylin and eosin sections by a registered neuropathologist (M.M.E. or Dr Catherine Joachim). Characteristics of the groups with and without SVD neuropathology are shown in Table 1. SVD was defined by vasculopathy-oriented criteria, as in our previous studies 2, 20 . These included: hyaline thickening of arteriolar walls; widened perivascular spaces; parenchymal changes considered to result from SVD (perivascular pallor of myelin staining, loosening with attenuation of nerve fibres with gliosis in white matter or loss of nerve cells and gliosis in deep grey matter) in one or more sections.

Human brain immunohistochemistry
A well-defined cohort of older individuals who had minimal AD pathology (Braak stage 0-II) were studied as in our previous report 20 , see Table 1. Frontal and parietal cortical tissue blocks including subcortical white matter were examined. Formalin-fixed paraffin embedded sections were immunohistochemically labelled as described previously 20 using a monoclonal antibody selective for type IV collagen, specifically for α1(IV) and α2(IV) collagens (clone COL-94, mouse IgG1, Sigma-Aldrich, Poole, UK).
Paraffin wax embedded sections (6 µm thickness) were processed for immunohistochemistry as in our previous studies 20 . Endogenous peroxidase activity was blocked by exposure to H2O2 (3% v/v, aqueous solution) for 8 min. After high-pressure heat-induced antigen retrieval (30 s, 125 o C, in pH 7.8 Tris-citrate buffer), non-specific binding was blocked with phosphate buffered saline containing 0.1 % v/v Triton-X100 and 3 % (w/v) bovine serum albumin (PBT-BSA) for 60 min at room temperature. Sections were then exposed to primary antibodies at 4 o C overnight.

Assessment of collagen-IV positive area fraction and sclerotic index
Sections were viewed on a Nikon Eclipse Ni-E Upright microscope with 20x or 40x objective lens. All vessels of arterial appearance within subcortical white matter in the size range 40 -150 μm least outer diameter were digitally sampled as TIFF files. To estimate the collagen-IV positive area fraction of each vessel wall, TIFF files were opened in Fiji-ImageJ software (https://imagej.net/Fiji). The background and lumen contents were removed (an example is shown in Figure 1, in the main document). Each image underwent colour deconvolution using the H-DAB macro within FiJi software, then a threshold detection algorithm within FiJi software was applied, using the default filter, to identify all collagen-IV positive pixels within the vessel wall ( Figure 1F, main document). Collagen-IV positive area fraction (AF, as %) was calculated as AF = 100 x (collagen-IV positive vessel wall area/total vessel wall area) for each vessel. Mean AF was calculated from all vessels meeting the inclusion criteria for each case.
Sclerotic index was estimated as a measure of vessel wall thickening, using the same TIFF files. On inspection, all small arteries with non-inflected circular cross section were selected. Vessels with squat ellipsoid cross section were also included, defined by greatest diameter less than 2x smallest diameter. For all selected vessels, the inner diameter and outer diameter were measured along the line of smallest inner diameter. Sclerotic index was computed as 1 -(inner diameter/outer diameter) and mean sclerotic index recorded for each case.
Harvesting of TIFF files and all image analyses were performed blind to clinical data.

Non-Human Primate model
Brain tissue from 17 male rhesus monkeys Macaca mulatta was used in this study 24 . This tissue had been collected and stored as part of a NIA-funded Program Project ("Neural Substrates of Cognitive Decline in Aging Monkeys," P01-AG000001) and a NINDS-funded Program Project ("Cognition and Cerebrovascular Disease," P01-NS031649). Monkeys were housed in the Laboratory Animal Science Center of Boston University, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. All animals were treated with strict accordance to the standards of the NIH Guide for the Care and Use of Laboratory Animals. Only male animals were studied to avoid confounding protective effects due to the female sex cycle. No unexpected mortality was seen in this cohort prior to planned sacrifice. Archival brain tissue was used from adult male M. mulatta that had been randomised either to undergo aortic narrowing to induce chronic severe hypertension or to remain unoperated (normotensive controls). Chronic hypertension was produced by surgically coarcting the aorta, details of which have been previously described 24 . Briefly, a 1-cm segment of thoracic aorta, at a level distal to the origin of the left subclavian artery, was narrowed to a luminal diameter of 2.0-3.0 mm. The coarctation of the aorta resulted in a decrease in luminal area of about 80%, as indicated by autopsy findings. The normotensive animals were unoperated controls. Though formal sample size calculations were not conducted, group sizes were based on a priori determined outcomes and previous studies from the present group 24 .
MRI scans were acquired using a Philips 3 Tesla scanner located in the Center for Biomedical Imaging at Boston University School of Medicine. Animals were initially anesthetized with ketamine hydrochloride (10 mg/kg, i.m.) and xylazine (1.25 mg/kg, i.m.) prior to MRI scanning. Supplemental doses of ketamine hydrochloride were then given as necessary to maintain a sufficient level of anesthesia.
Collagen-IV immunolabelled sections were scanned on a Nanozoomer slide scanner (Hamamatsu, UK) with 20x objective lens. Images were sampled as TIFF files in a stereotypical fashion from subcortical white matter areas. Tears, folds and other artefacts in the section were avoided. To estimate area fraction of collagen-IV immunoreactivity, TIFF files were imported into ImageJ software (http://rsb.info.nih.gov/ij/) and an automated threshold applied using a colour deconvolution plug-in. DAB-labelled area was determined, and collagen-IV-positive area fraction as a percent of the total area of the tiff file was calculated. Image analyses were performed blind to age or hypertensive status.

Hypertensive and normotensive rats
Rat studies were approved by the University of Glasgow Ethical Review Panel and complied with the Animals (Scientific Procedures) Act 1986 under Project Licence PPL-60/4286. Inbred colonies of the stroke-prone spontaneously hypertensive strain (SHRSP) and Wistar Kyoto (WKY) parent strain were maintained in the University of Glasgow by brother-sister mating. Maintenance of the colony integrity as well as the hypertensive and normotensive phenotypes was achieved by brother x sister mating, selecting SHRSP adult breeders with average systolic blood pressure 170-190 mmHg (males) and 130-150 mmHg (females), and WKY adult breeders with blood pressure <140mmHg (males) and <130mmHg (females) measured by tail-cuff plethysmography at age 12 weeks. Formal sample size calculations were not conducted, and group sizes were based on a priori determined outcomes and previous studies from the present group 19 . Only male animals were studied to avoid confounding protective effects due to the female sex cycle.
For this study male rats aged ≥10 months were deeply anaesthetised with 1.5-2.5 % isoflurane in a mixture of N2O/O2 (70/30) then heparinised saline was introduced transcardially, followed by 4 % paraformaldehyde (PFA) in phosphate-buffered saline (at least 200 ml, 80-100 mmHg perfusion pressure). Brains, while still in the skull, were postfixed in 4% PFA for 24 hours then removed and post-fixed for a further 24 hours. The brains were then cut into coronal blocks of 5 mm thickness and wax-embedded under vacuum. Adjacent sections (10 μm) were stained with haematoxylin and eosin (H&E) or with Masson trichrome stain.
For immunohistochemical labelling sections were processed as in our previous report 19 . Sections underwent heat-induced antigen retrieval (120 o C, 0.5 min) and blocking for 60 minutes at room temperature with 3 % (w/v) bovine serum albumen in phosphate-buffered saline containing 0.1 % Triton-X100 (PBT-BSA). They were then exposed to primary antibody to collagen type IV (rabbit polyclonal; Rockland Immunochemicals, Gilbertsville, PA) diluted 1:300 in PBT-BSA overnight at 4 o C. Immunolabelling was visualised using a peroxidise-conjugated secondary reagent and diaminobenzidine (DAB) chromagen (Envision® kit, Dako, Ely, UK), then lightly counterstained with Mayer's haematoxylin. As negative controls, matched sections treated identically but with irrelevant primary antibody (rabbit polyclonal anti-sheep IgG).
Sections were examined on a Zeiss Axioplan-2 microscope driven by Axiovision software (version 4.7). To study blood vessel morphometry a single blinded observer (P.A.) sampled all intact large leptomeningeal vessels at the ventral surface of the brain adjacent to the optic tract in coronal sections 0.5-1.5 mm anterior to bregma. To determine sclerotic index (S.I.=1 inner diameter/outer diameter) as a measure of vessel wall thickening, vessels with a noninflected circular or squat elliptical cross-sectional profile were sampled. To estimate area fraction of collagen-IV immunoreactivity within leptomeningeal vessels, images sampled as tiff files were imported into ImageJ software (http://rsb.info.nih.gov/ij/) and an automated threshold applied using a colour deconvolution plug-in. Inner and outer borders of vessel profile were delineated using a cursor and wall cross-sectional area estimated. DAB-labelled area within the vessel wall was determined, and collagen-IV-positive area fraction calculated. Image analyses were performed blind to hypertensive status.

Immuno-electron microscopy.
Thin sections of human brain were immunogold labelled for collagens I, III and IV (Rockland Immunochemicals, Gilbertsville, PA) as in our previous work 23 . Human cerebral cortical tissue from the superior frontal gyrus was sampled from formalin-fixed brains (n=6, ages: 46, 49, 51, 55, 62 and 71 years). These were processed for routine electron microscopy and post embedding immunogold EM. Briefly, small pieces (approx.1 mm 3 ) of tissues were dehydrated in 30%, 50%, 70%, and 90% EtOH, infiltrated, and embedded in LR White resin (Polysciences, Warrington, PA). Ultrathin sections collected on Formvar-coated nickel grids were incubated in primary antibodies overnight at 4°C, followed by secondary antibodies conjugated with 10 nm colloidal gold particles (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA, USA). Sections were stained briefly with uranyl acetate and lead citrate before examination with a Philips 208S electron microscope.   Immunolabelling is visualised with DAB chromogen (brown). Haematoxylin nuclear counterstain (blue). Scale bars: 20 μm.

Figure S3. Cerebral arterial fibrosis in chronically hypertensive rats.
A-D: leptomeningeal arteries of chronically-hypertensive rats (male SHRSP strain, age 10-12 months) and normotensive age-matched male normotensive rats of the parent strain (Wistar-Kyoto, WKY). Relative to normotensive rats (panel A) the hypertensive animals exhibited pronounced fibrosis in the arterial vessel wall, seen with the blue trichrome stain (B). Collagen-IV immunolabelling (brown) was evident as part of extracellular matrix throughout the artery wall in normotensive (panel C) and hypertensive (D) rats. Scale bars: 100 µm (A, B), 20 µm (C, D). E, F: sclerotic index (E) and extent of vascular collagen-IV immunolabelling (F) were quantified in leptomeningeal arteries from normotensive (Nor) WKY and hypertensive (Hyp) SHRSP rats (n=4, 7, respectively). Open blue symbols represent individual animals, filled orange symbols show group means. The arterial sclerotic index was higher in hypertensive SHRSP relative to normotensive WKY rats (P=0.038, Mann-Whitney U-test) in accord with our previous report{Brittain, 2013 #1239}. The extent of vascular collagen-IV immunolabelling in these vessels was not significantly different (P=0.762, Mann-Whitney Utest).