Hydralazine modulates expression of genes in the brain
Bulk RNA sequencing was performed on the brains of mice infused with vehicle, angiotensin II or angiotensin II + hydralazine. When comparing mice infused with angiotensin II alone versus vehicle alone, there were 1193 differentially expressed genes in the whole brain (687 upregulated and 506 downregulated; Figure 6A). There were 751 differentially expressed genes (327 upregulated and 424 downregulated) when comparing mice infused with angiotensin II alone versus angiotensin II + hydralazine in the whole brain (Figure 6B).
We next performed further analysis focussing on genes related to inflammation. In this analysis, the genes most upregulated by angiotensin II included cd163 molecule like 1 (Cd163l1 ), growth differentiation factor 7 (Gdf7 ), e74 like ETS transcription factor 3 (Elf3 ), cd3d delta subunit of t cell receptor complex (Cd3d ), cd209 molecule (Cd209 ), fibroblast factor growth factor 23 (Fgf23 ), interleukin 6 (Il6 ), t cell receptor beta joining 2-3 (Trbjj2-3) , histocompatibility 2, Q region locus 6 (H2-q6 ), z-dna binding protein 1 (Zbp1 ), coagulation factor XII (F12 ), CC motif chemokine ligand 8 (Ccl8 ), hematopoietic SH2 domain containing (Hsh2d ) and CC motif chemokine receptor 1 (Ccr1 ) (Figure 6C). The top downregulated genes following infusion with angiotensin II compared to vehicle included early growth response 2 (Egr2 ), fos proto-oncogene, AP-1 transcription factor subunit (Fos ) activity regulated cytoskeleton associated protein (Arc ), complement c7 (C7 ), BTG anti-proliferation factor 2 (Btg2 ), jun B proto-oncogene (Junb ), nuclear receptor subfamily 4 group A member (Nr4a1 ), dual specificity phosphatase 1 (Dusp1 ) and complement receptor 2 (Cr2 ) (Figure 6C).
The genes most upregulated by angiotensin + hydralazine compared to angiotensin II in the brain included endothelin 2 (Edn2), T cell receptor delta variable 1 (Trdv1 ), ubiquitin D (Ubd),interleukin 23 receptor (Il23r), toll like receptor 8(Tlr8), tripartite motif containing 38 (Trim38), C-C motif chemokine ligand 11 (Ccl11 ) and lymphocyte antigen 6 family member K (Ly6k ) (Figure 6D). The top downregulated genes by angiotensin II + hydralazine compared to angiotensin II in the brain included aquaporin-2 (Aqp2 ), uromodulin (Umod ), polymeric immunoglobulin receptor (Pigr ), sialic acid binding Ig like lectin 10 (Siglecg ), Sh2 domain containing 2a (Sh2d2a ), C-C motif chemokine ligand 8 (Ccl8 ), lymphocyte antigen 6 family member G (Ly6g) , interleukin 22 receptor subunit alpha1 (Il22ra ) and C-type lectin domain family 4 member D (Clec4d ) (Figure 6D).
When comparing mice infused with angiotensin II alone versus vehicle alone, there were 633 differentially expressed genes (324 upregulated and 309 downregulated) specifically in the hippocampus (Figure 7A). There were 921 differentially expressed genes (347 upregulated and 574 downregulated) in the hippocampus when comparing mice infused with angiotensin II alone versus angiotensin II + hydralazine (Figure 7B).
Further analysis revealed that the genes most upregulated by angiotensin II compared to vehicle in the hippocampus included interleukin 12 receptor subunit beta 2 (Il12rb2 ), protocadherin gamma subfamily b4 (Pcdhgb4 ), interferon induced transmembrane protein 7 (Ifitm7 ), hyaluronidase 1 (Hyal1 ), immunoglobulin superfamily member 6 (Igsf6 ), intercellular adhesion molecule 1 (Icam1 ), interleukin 2 receptor subunit beta (Il2rb ), annexin A11 (Anxa11 ), lysine demethylase 4a (Kdm4a ), TNF receptor superfamily member 25 (Tnfrsf25 ), docking protein 3 (Dok3 ) and B cell CLL/lymphoma 9 protein (Bcl9 ) (Figure 7C). The top downregulated genes following infusion with angiotensin II compared to vehicle included pterin-4 alpha-carbinolamine dehydratase (Pcbd1 ), angiotensinogen (Agt ), odd-skipped related transcription factor 1 (Osr1 ), formyl peptide receptor 1 (Fpr1 ), WD repeat domain 62 (Wdr62 ), FKBP prolyl isomerase 11 (Fkbp11 ), interleukin 1 receptor accessory protein 2 (Il1rapl2 ), macrophage stimulating 1 receptor (Mst1r ), tachykinin receptor 1 (Tacr1 ), natriuretic peptide A (Nppa ) and BarH like homeobox 1 (Barhl1 ).
The genes most upregulated by angiotensin + hydralazine compared to angiotensin II in the hippocampus included ninjurin 2 (Ninj2 ), wnt family member 4 (Wnt4 ), prostaglandin-endoperoxide synthase 1 (Ptgs1 ), interleukin-18 binding protein (Il18bp ), cd74 molecule (Cd74 ), tumour necrosis factor alpha-induced protein 2 (Tnfaip2 ), cathepsin k (Ctsk ), dual specificity phosphatase 10 (Dusp10 ), platelet factor 4 (Pf4 ), tripartite motif containing 4 (Trim4i ) and interleukin 1 receptor associated kinase 4 (Irak4 ) (Figure 7D). The top downregulated genes by angiotensin II + hydralazine compared to angiotensin II in the hippocampus included raf-1 proto-oncogene, serine/threonine kinase (Raf1 ), KLF transcription factor 9 (Klf9 ), protocadherin gamma subfamily A, 2 (Pcdhga2 ), C-X-C motif chemokine ligand 10 (Cxcl10 ), aquaporin 1 (Aqp1 ), ubiquitin specific peptidase 33 (Usp33 ), hyaluronidase 1 (Hyal1 ), midline 1 (Mid1 ), protocadherin gamma subfamily b4 (Pcdhgb4 ), diacylglycerol kinase gamma (Dgkg ), C-type lectin domain containing 10a (Clec10a ), interleukin 12 receptor subunit beta 2 (Il12rb2 ) and collagen type XIX alpha 1 chain (Col19a1 ).