Cholesterol impairs CaV1.2-mediated cerebral arteriolar contractility in humanized ApoE4 knock-in mice

The E4 allele of apolipoprotein E (ApoE4) is a significant genetic risk factor for cerebral small vessel disease (CSVD), a pervasive disorder characterized by damage to the cerebral microcirculation. In the brain, ApoE is primarily expressed in astrocytes and arteriolar vascular smooth muscle cells (aVSMCs). Expression of ApoE4 is known to disrupt lipid metabolism and transport, leading to elevated cellular cholesterol levels and contributing to glial and neuronal dysfunction. Notably, cholesterol can modulate ion channel activity, including L-type voltage-gated calcium channels (CaV1.2), which are highly expressed in aVSMCs and necessary for vascular contractility and autoregulation. Loss of autoregulation is a hallmark of CSVD, however whether cholesterol-dependent autoregulatory impairments underlie CSVD risk in ApoE4 carriers remains unknown. Therefore, we hypothesize that ApoE4-mediated increases in membrane cholesterol in cerebral arteriolar aVSMC leads to a reduction in CaV1.2 activity, thus impairing cerebral autoregulation. To test this hypothesis, freshly isolated cerebral aVSMC from 6-8 months old male humanized ApoE4 knock-in (hApoE4) and ApoE3 knock-in (hApoE3) mice were used for live-cell imaging of cholesterol with the cholesterol-sensitive dye Filipin-III (50 µg / mL). Additionally, peak amplitude of CaV1.2 currents was measured in aVSMCs using whole-cell patch clamp electrophysiology. Using pressure myography, we assessed the contractile response of pressurized cerebral arterioles to increasing intraluminal pressures (5 mmHg – 140 mmHg) and CaV1.2 channel activation with FPL 64176 (300 nM). Cellular cholesterol measurements in the perimeter of isolated aVSMC show that cells from hApoE4 mice have higher Filipin-III mean fluorescence intensity than those from hApoE3 controls. Further, CaV1.2 currents were significantly reduced in aVSMC of hApoE4 mice compared to hApoE3. Acute cholesterol depletion with methyl-β-cyclodextrin (MβCD, 5 mM) increased CaV1.2 currents in aVSMC of hApoE4, without any effects on hApoE3. Conversely, cholesterol-loading (1 mg/mL cholesterol) significantly decreased CaV1.2 currents in hApoE3 aVSMCs, but it did not further reduce the same currents in aVSMC from hApoE4. Consistent with these observations, pressurized cerebral arterioles from hApoE4 mice showed diminished contractile responses to FPL 64176 and blunted myogenic responses to stepwise increases in intraluminal pressure; both were restored by cholesterol removal with MβCD. In summary, these findings suggest that elevated membrane cholesterol in hApoE4 cerebral aVSMCs blunts CaV1.2 channel activity, leading to impaired arteriolar contractility, which is restored by cholesterol depletion. These observations offer mechanistic insight into ApoE4-related alterations in cerebral aVSMC function, potentially linking ApoE4 expression to the pathophysiology of vascular dysfunction in CSVD. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.