Inflammatory pathways are central to posterior cerebrovascular artery remodelling prior to the onset of congenital hypertension

Cerebral artery hypoperfusion may provide the basis for linking ischemic stroke with hypertension. Brain hypoperfusion may induce hypertension that may serve as an auto-protective mechanism to prevent ischemic stroke. We hypothesised that hypertension is caused by remodelling of the cerebral arteries, which is triggered by inflammation. We used a congenital rat model of hypertension and examined age-related changes in gene expression of the cerebral arteries using RNA sequencing. Prior to hypertension, we found changes in signalling pathways associated with the immune system and fibrosis. Validation studies using second harmonics generation microscopy revealed upregulation of collagen type I and IV in both tunica externa and media. These changes in the extracellular matrix of cerebral arteries pre-empted hypertension accounting for their increased stiffness and resistance, both potentially conducive to stroke. These data indicate that inflammatory driven cerebral artery remodelling occurs prior to the onset of hypertension and may be a trigger elevating systemic blood pressure in genetically programmed hypertension.

• The outer collagen layer • The vessel wall • The total vessel size (including collagen and lumen) Automatic measurements of size and shape of these masks were performed in ImageJ. The masks were also applied to the original maximum intensity image to make measurements of the picrosirius red staining. All measurements were outputted to the results/summary window in ImageJ and then further analysis.

a)
Maximum intensity projection of backward scattered SHG signal b) User asked to select outer edge of vessel. Binary mask created. c) User asked to select adventitia. Binary mask created d) Binary mask c subtracted from binary mask b. e) Intensity measurements made using the resultant binary masks on original data a.

Figure S2. Panther database gene ontology analysis of differentially expressed genes
The genes presented are differentially transcribed between aged matched WKY and SHR and categorized based on their A) protein class and B) the molecular function they play.   Figure S3.

Ingenuity pathway analysis of diseases and biofunctions that the differentially expressed genes might be involved in at various stage of hypertension in the SHR
A maximum of 20 biofunctions with highest probability are shown for each age. The p values reported are false discovery rate corrected (Benjamini-Hochberg correction). These biofunctions are highlighted based on pairwise comparison of differential gene expression between SHR and age matched WKY. The common biofunction across all ages analysed are listed in Table S1. The dotted line represents the threshold value of p=0.05.

Table S1. Biofunctions highlighted in all age matched conditions
The probable biofunctions that are affected by differential gene expression between WKY and SHR. These biofunctions are common to all analysed ages. The probabilities reported are Benjamini-Hochberg corrected. Chitinase-3-like protein 1; Carbohydrate-binding lectin with a preference for chitin. Has no chitinase activity. May play a role in tissue remodelling and in the capacity of cells to respond to and cope with changes in their environment. Plays a role in T-helper cell type 2 (Th2) inflammatory response and IL-13-induced inflammation, regulating allergen sensitization, inflammatory cell apoptosis, dendritic cell accumulation and M2 macrophage differentiation.

Cxcl10
-0.75 0.07 C-X-C motif chemokine 10 ; In addition to its role as a proinflammatory cytokine, may participate in T-cell effector function and perhaps T-cell development Cxcr4 0.52 0.06 C-X-C chemokine receptor type 4 ; Receptor for the C-X-C chemokine CXCL12/SDF-1 that transduces a signal by increasing intracellular calcium ion levels and enhancing MAPK1/MAPK3 activation. Acts as a receptor for extracellular ubiquitin; leading to enhanced intracellular calcium ions and reduced cellular cAMP levels. Involved in haematopoiesis and in cardiac ventricular septum formation. Also plays an essential role in vascularization of the gastrointestinal tract, probably by regulating vascular branching and/or remodelling processes in endothelial cells.

F2rl1
-1.26 0.002 Proteinase-activated receptor 2 ; Receptor for trypsin and trypsin-like enzymes coupled to G proteins. Its function is mediated through the activation of several signalling pathways including phospholipase C (PLC), intracellular calcium, mitogen-activated protein kinase (MAPK), I-kappaB kinase/NF-kappaB and Rho. Can also be transactivated by cleaved F2r/Par1. Involved in modulation of inflammatory responses and regulation of innate and adaptive immunity, and acts as a sensor for proteolytic enzymes generated during infection. Generally, is promoting inflammation. Oxidized low-density lipoprotein receptor 1 Oxidized low-density lipoprotein receptor 1, soluble form; Receptor that mediates the recognition, internalization and degradation of oxidatively modified low density lipoprotein (oxLDL) by vascular endothelial cells. OxLDL is a marker of atherosclerosis that induces vascular endothelial cell activation and dysfunction, resulting in pro-inflammatory responses, pro-oxidative conditions and apoptosis. Its association with oxLDL induces the activation of NF-kappa-B through an increased production of intracellular reactive oxygen species Prg2 -1.08 0.018 Bone marrow proteoglycan precursor; Cytotoxin and helminthotoxin. MBP also induces non-cytolytic histamine release from basophils. It is involved in antiparasitic defence mechanisms and immune hypersensitivity reactions RT1-Bb -0.62 0.004 Rano class II histocompatibility antigen, B-1 beta chain precursor; Involved in the presentation of foreign antigens to the immune system RT1-Db1 -0.84 0.002 Rano class II histocompatibility antigen, D-1 beta chain precursor Tgfb3 0.45 0.014 Transforming growth factor beta-3; Involved in embryogenesis and cell differentiation 5 Table S3. Transcripts involved in immune system signalling -receptors and ligands. Genes that transcript changed in the pre hypertensive (5 6 weeks old) SHR as compared to aged matched WKY