Masayo Koide

Brain capillaries sense neural activity and direct blood flow to active regions-a process termed neurovascular coupling that underlies activity-dependent increases in local perfusion (functional hyperemia). A key contributor to functional hyperemic responses is the capillary endothelial cell (cEC) inward rectifier K⁺ (Kir2.1) channel, which, when activated by neuronal activity-derived extracellular K⁺, initiates vasodilatory electrical signals that propagate through the vascular network. Kir2.1 channel function requires continual production of its lipid cofactor, phosphatidylinositol-4,5-bisphosphate (PIP₂), and is compromised in mouse models of cerebral small vessel (cSVD). Although decreased PIP₂ availability is a common feature of cSVDs, mechanisms underlying PIP₂ synthesis remain poorly understood. We hypothesized that Arf6, a small GTPase expressed in cECs that stimulates PIP₂ production, is critical for this process. Using patch-clamp electrophysiology, we demonstrate that inhibiting Arf6 activity progressively decreased cEC Kir2.1 channel activity. This deficit manifested as loss of capillary-to-arteriole electrical signaling in isolated vessels and diminished functional hyperemia in vivo. Exogenously provided PIP₂ restored Kir2.1 currents and functional hyperemia after Arf6 inhibition or genetic knockdown. Collectively, our data suggest that cEC Arf6 sustains Kir2.1 activity by maintaining PIP₂ levels and demonstrate that diminished PIP₂ synthesis is sufficient to impair functional hyperemia. Furthermore, we identify Arf6 as a mechanistic link between PIP₂ production and endothelial electrical signaling, highlighting Arf6 as a potential therapeutic target for restoring functional hyperemia.

Brain capillaries sense neural activity and direct blood flow to active regions—a process termed neurovascular coupling that underlies activity-dependent increases in local perfusion (functional hyperemia). A key contributor to functional hyperemic responses is the capillary endothelial cell (cEC) inward rectifier K+ (Kir2.1) channel, which is activated by neuronal activity-derived extracellular K ⁺ and initiates vasodilatory electrical signals that propagate through the vascular network. Kir2.1 channel function requires continual production of its lipid cofactor, phosphatidylinositol-4,5-bisphosphate (PIP ₂ ), and is compromised in mouse models of cerebral small vessel (cSVD). Although decreased PIP ₂ availability is a common feature of cSVD, mechanisms underlying PIP ₂ synthesis remain poorly understood. We hypothesized that Arf6, a small GTPase expressed in cECs that stimulates PIP ₂ production, is critical for this process. Using patch-clamp electrophysiology, we demonstrate that inhibiting Arf6 activity progressively decreased cEC Kir2.1 channel activity. This deficit corresponded to loss of capillary-to-arteriole electrical signaling in isolated vessels and diminished functional hyperemia in vivo. Exogenously provided PIP ₂ restored Kir2.1 currents and functional hyperemia after Arf6 inhibition or genetic knockdown. Collectively, our data indicate that cEC Arf6 sustains Kir2.1 activity by maintaining PIP ₂ levels and demonstrate that diminished PIP ₂ synthesis is sufficient to impair functional hyperemia. Furthermore, we identify Arf6 as a mechanistic link between PIP ₂ production and endothelial electrical signaling, highlighting Arf6 as a potential therapeutic target for restoring functional hyperemia.

Functional hyperemia – activity-dependent increases in local blood perfusion – underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by exogenous administration of PIP ₂ , but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs depletes PIP ₂ and attenuates functional hyperemia, underscoring the central role of the endothelial EGFR signaling in cerebral blood flow regulation.

The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary inositol 1,4,5-triphosphate receptor (IP ₃ R)–mediated calcium (Ca ²⁺ ) signals to direct blood flow. The initiation and amplification of endothelial cell IP ₃ R-mediated Ca ²⁺ signals requires an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial IP ₃ -evoked Ca ²⁺ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca ²⁺ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca ²⁺ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca ²⁺ activity and cortical capillary blood flow in vivo within 120 seconds. Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca ²⁺ signaling and endothelium-dependent vasodilation.

The dense network of capillaries composed of capillary endothelial cells (cECs) and pericytes lies in close proximity to all neurons, ideally positioning it to sense neuron- and glial-derived compounds that enhance regional and global cerebral perfusion. The membrane potential ( V _M ) of vascular cells serves as the physiological bridge that translates brain activity into vascular function. In other beds, the ATP-sensitive K ⁺ (K _ATP ) channel regulates V _M in vascular smooth muscle, which is absent in the capillary network. Here, with transgenic mice that expressed a dominant-negative mutant of the pore-forming Kir6.1 subunit specifically in brain cECs or pericytes, we demonstrated that K _ATP channels were present in both cell types and robustly controlled V _M . We further showed that the signaling nucleotide adenosine acted through A _2A receptors and the Gα _s /cAMP/PKA pathway to activate capillary K _ATP channels. Moreover, K _ATP channel stimulation in vivo increased cerebral blood flow (CBF), an effect that was blunted by expression of the dominant-negative Kir6.1 mutant in either capillary cell type. These findings establish an important role for K _ATP channels in cECs and pericytes in the regulation of CBF.

Cerebral blood flow (CBF) is exquisitely controlled to meet the ever-changing demands of active neurons in the brain. Brain capillaries are equipped with sensors of neurovascular coupling agents released from neurons/astrocytes onto the outer wall of a capillary. While capillaries can translate external signals into electrical and Ca²⁺ changes, control mechanisms from the lumen are less clear. The continuous flux of red blood cells and plasma through narrow-diameter capillaries imposes mechanical forces on the luminal (inner) capillary wall. Whether—and, if so, how—the ever-changing CBF could be mechanically sensed in capillaries is not known. Here, we propose and provide evidence that the mechanosensitive Piezo1 channels operate as mechanosensors in CNS capillaries to ultimately regulate CBF. Patch clamp electrophysiology confirmed the expression and function of Piezo1 channels in brain cortical and retinal capillary endothelial cells. Mechanical or pharmacological activation of Piezo1 channels evoked currents that were sensitive to Piezo1 channel blockers. Using genetically encoded Ca²⁺ indicator (Cdh5-GCaMP8) mice, we observed that Piezo1 channel activation triggered Ca²⁺ signals in endothelial cells. An ex vivo pressurized retina preparation was employed to further explore the mechanosensitivity of capillary Piezo1-mediated Ca²⁺ signals. Genetic and pharmacologic manipulation of Piezo1 in endothelial cells had significant impacts on CBF, reemphasizing the crucial role of mechanosensation in blood flow control. In conclusion, this study shows that Piezo1 channels act as mechanosensors in capillaries, and that these channels initiate crucial Ca²⁺ signals. We further show that Piezo1 modulates CBF, an observation of profound significance for the control of brain blood flow in health and in disorders where hemodynamic forces are disrupted, such as hypertension.