Epidermal growth factor receptors in vascular endothelial cells contribute to functional hyperemia in the brain

Abstract

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 (PIP₂) 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 PIP₂, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs depletes PIP₂ and attenuates functional hyperemia, underscoring the central role of the endothelial EGFR signaling in cerebral blood flow regulation.

Keywords: Functional hyperemia; cerebral small vessel diseases (cSVD); epidermal growth factor receptor (EGFR); phosphatidylinositol 4,5-bisphosphate (PIP2); vascular endothelial cells.

Figure 1:

Characterization of capillary ECs from EC-EGFR-KO mice. A: Bright field and fluorescent images of isolated microvessels from EC/SMC dual-reporter mice. Green fluorescent protein expresses in ECs under the cadherin-5 (cdh5) promotor, and red fluorescent protein expresses in SMC/pericytes under the acta2 promoter. The vast majority (>98%) of tissue obtained was green fluorescent-positive and red fluorescent-negative microvessels, demonstrating that the collected tissue samples were capillaries. B: EGFR protein in freshly isolated capillaries, pooled from 4 animals for each sample, were quantified by ELISA. EGFR protein was dramatically decreased in EC-EGFR-KO mice compared to WT animals (i.e., Cre-negative littermates). Data are presented as mean ± SEM (n = 8 in WT, n = 6 in KO). *** P < 0.001 between groups by unpaired t-test.

Figure 2:

Impaired functional hyperemia in EC-EGFR-KO mice. A: Representative traces showing CBF increases in contralateral somatosensory cortex during whisker stimulation. Cre-negative littermates (WT) were used as a control group in comparison to EC-EGFR-KO (KO) mice. B: Summary data of whisker stimulation-induced functional hyperemia. C: CBF increase during whisker stimulation after treatment with Ba²⁺ (100 μM), a Kir2.1 channel blocker. D: Ba²⁺ sensitive-component of functional hyperemia, i.e., the difference in responses before and after Ba²⁺ treatment, indicating the contribution of the Kir2.1 channels to whisker stimulation-induced functional hyperemia. Data are presented as mean ± SEM (n = 8 in WT, n = 8 in KO). **** P < 0.0001, *** P < 0.001 between groups by unpaired t-test.

Figure 3:

PIP₂, an endogenous Kir2.1 channel co-factor, restored functional hyperemia in EC-EGFR-KO mice. A: Whisker stimulation-induced functional hyperemia before and after PIP₂ treatment in EC-EGFR-KO mice. B: Summary data showing PIP₂ treatment restores functional hyperemia deficits in EC-EGFR-KO mice, with little impact on WT animals. C: Ba²⁺ sensitive-component of functional hyperemia after PIP₂ treatment. Data are presented as mean ± SEM (n = 4 in WT, n = 4 in KO). ** P < 0.01, ns: not significant between groups by paired t-test (B) or by unpaired t-test (C).

Figure 4:

HB-EGF, an EGFR ligand, failed to restore functional hyperemia in EC-EGFR-KO mice. A: Whisker stimulation-induced functional hyperemia before and after HB-EGF treatment in EC-EGFR-KO mice. B: Summary data showing that HB-EGF treatment did not alter whisker stimulation-induced functional hyperemia in either EC-EGFR-KO or WT mice. C: Ba²⁺ sensitive-component of functional hyperemia after HB-EGF treatment. Data are presented as mean ± SEM (n = 4 in WT, n = 4 in KO). * P < 0.05, ns: not significant between groups by paired t-test (B) or by unpaired t-test (C).

Figure 5:

Schematic illustration of the proposed signaling pathway underlying functional hyperemia deficits in EC-EGFR-KO mice. Whisker stimulation and subsequent neuronal activation, which increases perivascular K⁺ during neuronal repolarization, results in Kir2.1 channel activation in capillary ECs. Kir2.1 channel-initiated hyperpolarizing vasodilatory signal rapidly propagates upstream and dilates the precapillary arteriole, increasing downstream tissue perfusion. This local increase in blood flow in neuronally active regions of the brain is termed functional hyperemia, which is detected using laser Doppler flowmetry in this study. Genetic deletion of EGFR disrupts functional hyperemia in response to whisker stimulation by causing Kir2.1 channel dysfunction, which is likely attributed to lowering the level of PIP₂, an essential co-factor of Kir2.1 channels.