Differential Gene and Protein Expressions Responsible for Vasomotor Signaling Provide Mechanistic Bases for the Opposite Flow-Induced Responses of Pre- and Post-Circle of Willis Arteries

Figures

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  Figure 1. Protein levels of vasoconstrictor enzymes COX1/2, CYP4A and TP receptors are significantly lower in BA, while the protein level of TXAS is similar between BA and MCA arteries. Representative Western blots and group data of densitometric analysis of COX1 (A) and COX2 (B), TXAS (C), TP receptor (D), and CYP4A (E) levels are shown. Results were normalized to β-actin levels. Comparisons were made by parametric two-tailed independent t-tests. Data are expressed as mean ± SEM of three individual samples (six animals/sample) from 1–2 separate trials. p < 0.05 was considered statistically significant. BA—basilar artery; MCA—middle cerebral artery; COX1/2—cyclooxygenase 1 and 2; TP receptor/TBXA2R—thromboxane A2 receptor; TXAS—thromboxane synthase; CYP4A—Cytochrome P450 4A.

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  Figure 2. Protein levels of prostacyclin synthase and prostacyclin receptor, as well as neural and endothelial NOS enzymes in BA and MCA. The levels of PGIS and PTGIR proteins are significantly higher in BA than MCA (A,B). The levels of nNOS and eNOS proteins are similar in BA and MCA (C,D). Representative Western blots and group data of densitometric analysis of PGIS (A), PTGIR (B), neural NOS (C), and endothelial NOS (D) levels are shown. The results were normalized to β-actin levels. Comparisons were made by parametric two-tailed independent t-tests. Data are expressed as mean ± SEM of three individual samples (six animals/sample) from 1–2 separate trials. p < 0.05 was considered statistically significant. BA—basilar artery; MCA—middle cerebral artery; PGIS—prostacyclin synthase; PTGIR—prostacyclin receptor; nNOS—neural nitric oxide synthase; eNOS—endothelial nitric oxide synthase.

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  Figure 3. Volcano plot demonstrating 636 differentially expressed genes (DEGs) in cerebral arteries of anterior (CAACC) relative to posterior (CAPCC). Ingenuity Pathway Analysis (IPA) identified 362 differentially expressed genes (DEGs) that were upregulated and 274 DEGs that were downregulated in CAACC relative to CAPCC. The light green dots represent downregulated genes (FC < −1.5), while the red dots represent upregulated genes (FC > 1.5) in CAACC relative to CAPCC. The threshold of the FDR-adjusted p-value was set <0.05.

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  Figure 4. Ingenuity pathway analysis (IPA) of differentially expressed genes of canonical pathways involved in vasoconstriction (A) and vasodilation (B) in CAACC relative to CAPCC. Selected canonical pathways are grouped by their proposed effect on vascular tone regulation and presented accordingly. Adjusted p-values of pathway enrichment are visualized on a −(lg) scale, where the values above 1.3 equal the threshold of significance below 0.05. The ratio value is calculated by dividing the number of DEGs mapped to the pathway by the number of all the genes present on the given canonical pathway. The coloring of the bars represents the predicted activity of the canonical pathways calculated as Z-scores. DEGs—differentially expressed genes; CAACC—cerebral arteries of the anterior cerebral circulation; CAPCC—cerebral arteries of the posterior cerebral circulation.

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  Figure 5. Illustration of the anatomical origin of arteries investigated in the present study. The important point is that on the base of the brain, the circle of Willis is located, is supplied by several arteries, and serves to equalize the pressure and flow in the circle of Willis before further daughter arterial branches reach the intracerebral spaces. Because of this special arrangement, the circle of Willis can provide compensation and maintenance of blood supply to the anterior brain regions if one of the supplying arteries is stenotic or blocked. ICA—internal carotid artery; WSS—wall shear stress. The green line with an arrowhead indicates the direction of blood flow. The black line indicates blocked blood flow through the middle cerebral artery.

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  Figure 6. Proposed molecular mechanisms of the opposite vasomotor responses of the middle cerebral and basilar arteries to flow. An increase in intraluminal flow elicits the release of arachidonic acid (AA) from the cell membrane via activation of phospholipase A2 (PLA2). This flow elicits constriction in the internal carotid system, such as the middle cerebral artery (MCA), while in the vertebro-basilar system, such as the basilar artery (BA), the flow elicits dilation. We propose that different expressions of enzymes producing AA metabolites may be responsible for the opposite vasomotor responses. Expression of enzymes, thus the production of constrictor AA metabolites, is greater in the MCA than in BA, whereas expression of enzymes producing dilator AA metabolites is greater in BA than in MCA. Flow-induced constriction is mediated primarily by 20-HETE produced by cytochrome P450 4A (Cyp4A) enzymes and requires COX1/2 activity and thromboxane (TP) receptor, whereas the flow-induced dilation is mediated by dilator AA metabolites, such as prostacyclin (PGI2) produced by PGI2 synthase (PGIS) and the PGI2 receptor (IP). Nitric oxide (NO) produced by NO synthases (nNOS/eNOS) in response to…