As a leading cause of stroke and dementia, cerebral small vessel diseases (SVDs) pose a horrendous threat to the elderly population (Pantoni, 2010; Wardlaw et al., 2019). Despite their major contribution to age-related vascular cognitive impairment and disability (Iadecola et al., 2019), the disease processes and key biological mechanisms underlying these disorders remain largely unknown. Moreover, there are no specific treatments outside the management of vascular risk factors and use of anti-platelets after ischemic stroke. However, accumulating experimental evidence suggests that functional alterations in the cerebral microcirculation have early and deleterious consequences on functional hyperemia—the ability of the brain to increase local blood flow in response to local increases in neuronal activity (Huneau et al., 2018; Iadecola et al., 2019).

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common genetic form of cerebral SVDs. Remarkably, CADASIL includes all clinical and MRI manifestations of sporadic SVDs, hence offering a lens through which to view more common forms of sporadic SVDs (Chabriat et al., 2009). Both CADASIL patients and the TgNotch3^R169C mouse model, hereafter referred to as SVD mice, exhibit deficits in functional hyperemia at an early stage of the disease progression (Joutel et al., 2010; Huneau et al., 2018). We have recently demonstrated that activation of the epidermal growth factor receptor (EGFR) by its ligand heparin-binding EGF-like growth factor (HB-EGF) ameliorates the cerebral vascular deficits of the SVD mouse—including impaired functional hyperemia (Capone et al., 2016; Dabertrand et al., 2021). Part of this effect is mediated by reenabling capillary-to-arteriole signaling during the neurovascular coupling that underpins functional hyperemia (Dabertrand et al., 2021). In physiological conditions, action potentials increase extracellular K⁺ concentration which activates the strong inward-rectifier K⁺ (Kir2.1) channel in capillary endothelial cells (cECs). This creates a hyperpolarizing signal that rapidly propagates to upstream arterioles, driving vasodilation and local hyperemia (Longden et al., 2017; Harraz et al., 2018a; Moshkforoush et al., 2020). This mechanism is vulnerable to pathology, and we recently demonstrated that SVD lowers the synthesis of the phospholipid PIP₂, which prevents Kir2.1 channels from acting as sensors of increases in external K⁺ (Dabertrand et al., 2021). Strikingly, we showed that both HB-EGF and systemic injection of exogenous PIP₂ improved functional hyperemia deficits in SVD mice by restoring capillary-to-arteriole signaling.

Functional hyperemia is a vital process controlled by multiple mechanisms that provide layers of redundancy, and thus neurovascular coupling agents other than K⁺ ions have been postulated (Attwell et al., 2010; Kaplan et al., 2020). Among these, prostaglandin E₂ (PGE₂), produced by cyclooxygenase-2 (COX-2) from arachidonic acid, has been widely proposed to be released from excitatory neurons to relax arteriolar smooth muscle cells (SMCs) by activation of the G_S protein-coupled prostanoid EP4 receptor and subsequent cyclic adenosine monophosphate-dependent pathway (Zonta et al., 2002; Takano et al., 2005; Gordon et al., 2008; Attwell et al., 2010; Lacroix et al., 2015). Yet, this interpretation appears incompatible with our previous demonstration that PGE₂ causes constriction, rather than dilation, when applied directly to isolated cortical arterioles—an effect that occurs via activation of the G_q protein-coupled prostanoid EP1 receptor (Dabertrand et al., 2013). Consistent with these observations, the EP1 receptor is robustly expressed not only in the arteriolar SMCs, but also in cECs of the brain microcirculation (Vanlandewijck et al., 2018). We therefore hypothesized that PGE₂ contributes to neurovascular coupling by initiating a vasodilatory signal from the capillaries to the upstream parenchymal arterioles, rather than by targeting SMCs directly. Using a combination of ex vivo and in vivo approaches, we report that capillary stimulation with PGE₂ induces upstream arteriolar dilation and increases blood flow in vivo. Consistent with defective functional hyperemia, we further show that PGE₂-induced capillary-to-arteriole signaling is blunted in the SVD mouse model and can be rescued by HB-EGF.

To investigate the effect of capillary stimulation with PGE₂ on upstream arteriolar diameter, we used our previously developed ex vivo capillary-parenchymal arteriole (CaPA) preparation that allows to apply vasoactive substances at specific points along the arteriole-capillary continuum by pressure ejection (Longden et al., 2017; Rosehart et al., 2019). Consistent with our previous report that PGE₂ acts as a vasoconstrictor (Dabertrand et al., 2013), local application of 1 μM PGE₂ directly on the arteriolar segment caused a decrease in diameter (Figures 1A–C). In contrast, and as expected (Longden et al., 2017; Dabertrand et al., 2021), 10 mM K⁺ also applied on the arteriole caused a robust dilation. Interestingly, either 10 mM K⁺ or 1 μM PGE₂ caused upstream dilations when applied onto capillary extremities and the amplitudes of these were virtually identical (56.2% ± 3.9% and 47.8% ± 4.2%, respectively) (Figures 1A–C and Supplementary Movie 1). However, the kinetics of the responses appear significantly different with slower onset (5.27 ± 1.35 s) and time to peak (9.65 ± 1.5 s) for PGE₂ compared to K⁺ responses, (1.7 ± 0.4 s and 3.87 ± 1.13 s, respectively).

We next tested the effect of increasing concentrations of PGE₂ locally applied to the arteriolar segment and observed a concentration-dependent constriction with a calculated EC₅₀ of 145 nM (Figure 1D). The arteriolar dilation in response to capillary stimulations with PGE₂ was also concentration-dependent with a calculated EC₅₀ of 70 nM and a maximal response at 1 μM, and thus we chose to use the latter concentration throughout the study for capillary stimulation (Figure 1E and Supplementary Movie 1). Finally, we tested the effect of a modest concentration of PGE₂ (500 nM) applied via the bath perfusion, hence stimulating both the capillary ends and the arteriolar segment, and measured a small but clear constriction (Figures 1F,G).

In our initial report of capillary-to-arteriole electrical signaling (Longden et al., 2017), we identified the cEC strong inward-rectifier K⁺ channel, Kir2.1, as the molecular cornerstone of the capillary-to-arteriole electrical mechanism elicited by capillary stimulation with 10 mM K⁺. Increasing extracellular K⁺ concentration from 3 to 10 mM activates Kir2.1 channels in cECs, which induces a regenerative hyperpolarization that travels retrogradely to dilate the upstream arteriole. Consistent with this model, 30 μM Ba²⁺—which, among the inward rectifier K⁺ channels expressed in the microcirculation, preferentially blocks Kir2 channels (Longden and Nelson, 2015)—completely abolished the arteriolar dilation in response to capillary-applied 10 mM K⁺. Therefore, we tested the effect of Ba²⁺ on arteriolar dilation induced by capillary stimulation with PGE₂. Arteriolar diameter was recorded at two distinct zones: zone 1 was placed at the branching of the transitional segment from the arteriole and zone 2 was located 250 μm upstream of this (Figure 2A). PGE₂ locally applied onto capillaries led to similar dilatory responses in zone 1 (49.1% ± 5.5%) and zone 2 (48.2% ± 2.9%) (Figures 2B,C). Remarkably, bath application of 30 μM Ba²⁺ decreased upstream arteriolar dilation at zone 1 in response to capillary stimulation with PGE₂ by 63% and virtually eliminated dilation at zone 2. The onset of the remnant dilation observed at zone 1 was unchanged by Ba²⁺ (5.27 ± 1.35 s 6.49 ± 2.81 s). Taken together, these data suggest that the Kir2.1 channel is not required to initiate PGE₂-induced upstream arteriolar dilation, but rather participates in the amplification and propagation of the vasodilatory signal, as evidenced by the difference between zone 1 and zone 2.

To investigate the mechanistic underpinnings of the capillary-to-arteriole signaling induced by PGE₂, we first superfused the ex vivo CaPA preparation with 1 μM SC51322, a prostanoid receptor antagonist specific for the EP1 receptor. SC51322 abolished the response to PGE₂, while the dilation induced by 10 mM K⁺ remained intact (Figures 3A,B). This observation suggests that PGE₂ acts through activation of G protein-coupled receptors of the G_q/11 subtype (G_qPCR) and subsequent Ca²⁺ signaling. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) following G_qPCR activation is known to stimulate transient receptor potential (TRP) channels, a major pathway for extracellular Ca²⁺ influx (Kim et al., 2008; Harraz et al., 2018b). Moreover, recent work by Thakore et al. (2021) has revealed that activation of TRPA1 channel in cECs can initiate a biphasic, propagating retrograde signal that dilates upstream parenchymal arterioles during functional hyperemia. However, the TRPA1 antagonist HC030031 at 3 μM had no effect on the dilation induced by capillary stimulation with PGE₂ (Figures 3A,B). Inhibition of TRPV4, another Ca²⁺-permeable TRP channel expressed by cECs (Harraz et al., 2018b), with 1 μM HC067047 also did not impact the effect of PGE₂ (Figures 3A,B). These results suggest that PGE₂ induces upstream arteriole dilation via activation of the EP1 receptor but independently of TRPV4 and TRPA1 channels.

We then tested the ability of PGE₂ to dilate upstream arterioles in vivo by measuring capillary red blood cell (RBC) flux with two-photon laser-scanning microscopy. Fluorescein isothiocyanate (FITC)-labeled dextran was used to visualize the cortical microcirculation and a pipette containing 1 μM PGE₂ was maneuvered into the brain through a cranial window and positioned next to a capillary of interest (Figure 4A). Pressure ejection (8 ± 1 psi) for up to 300 ms of PGE₂ evoked a significant increase in capillary RBC flux (Δ = 12 ± 4 RBCs/s) consistent with the notion that the mechanisms we observed in CaPA preparations are also at play in the intact system (Figures 4B–D). In contrast, ejection of aCSF vehicle had no effect on capillary RBC flux (Figure 4E). To determine whether PGE₂ also causes upstream dilation we next performed experiments in which we imaged at the parenchymal arteriole-transitional zone junction (Figure 4F). Here, we found that ejection of 1 μM PGE₂ selectively onto capillaries routinely produced a small upstream dilation (Figures 4F,G and Supplementary Movie 2). In contrast, if we applied PGE₂ onto both capillaries and arterioles in vivo by increasing the duration of ejection, causing spread of the ejected solution to the upstream arteriole along a paravascular route, we observed constriction (Figure 4H), consistent with our ex vivo data. As expected, ejection of aCSF vehicle alone had no effect on upstream arteriolar diameter (12.95 ± 0.57 μm baseline diameter vs. 12.96 ± 0.8 μm after aCSF ejection, n = 6 experiments, 3 mice, P = 0.97, paired Student’s t-test).

Finally, we investigated the effect of SVD on PGE₂-initiated capillary-to-arteriole signaling. Ex vivo stimulation of capillaries from SVD mice with PGE₂ induced an upstream arteriolar dilation that was 58% smaller at Zone 1 and 64.8% smaller at Zone 2 compared with that in TgNotch3^WT control (WT) mice, revealing attenuated PGE₂-mediated signaling (Figure 5). The difference between zone 1 and 2 dilations were not significant. We previously reported that activation of the epidermal growth factor receptor (EGFR) by its ligand heparin-binding EGF-like growth factor (HB-EGF) ameliorates the cerebral vascular deficits of the SVD mouse—including neurovascular coupling and functional hyperemia deficits (Capone et al., 2016; Dabertrand et al., 2021). We then directly tested the effect of HB-EGF on PGE₂-induced upstream vasodilation in ex vivo preparations from SVD mice. Bath-applied 30 ng/mL HB-EGF dramatically enhanced the upstream arteriolar dilation induced by capillary stimulation with PGE₂, increasing the average dilation from 19.8% ± 1% to 48.7% ± 4.3% at Zone 1, and from 16.8% ± 1.4% to 45.3% ± 5.1% at Zone 2 (Figure 5 and Supplementary Movie 3). HB-EGF completely restored PGE₂-induced upstream dilation, abolishing the differences measured between WT and SVD animals.

Our progress in understanding functional hyperemia in health and disease has been hampered by large gaps in our comprehension of the mechanism underlying this basic physiological response—and persistent controversies surrounding it (Kaplan et al., 2020). Our recent work identified Kir2.1 channels in cECs as the molecular cornerstone initiating and propagating a retrograde hyperpolarizing vasodilatory signal from capillaries to arterioles (Longden et al., 2017; Harraz et al., 2018a; Moshkforoush et al., 2020; Dabertrand et al., 2021). The present study extends this capillary-based paradigm, providing support for a new signaling modality that posits a central role for the G_qPCR EP1 receptor in mediating PGE₂-induced vasodilatory signal that propagates upstream to cause dilation of feeding parenchymal arterioles (Figure 6). Importantly, these findings hold the promise of resolving controversies surrounding how PGE₂, a widely proposed mediator of neurovascular coupling (Zonta et al., 2002; Takano et al., 2005; Gordon et al., 2008; Attwell et al., 2010; Watkins et al., 2014), can promote functional hyperemia despite evidence that it directly constricts arterioles (Dabertrand et al., 2013).

Our observations raise the immediate question of which effect of PGE₂, capillary-mediated vasodilation or direct arteriolar constriction, predominates during neurovascular coupling. We calculated an EC₅₀ of 145 nM for the PGE₂-induced constriction of the parenchymal arteriole, while the vasodilation induced by capillary stimulation displayed an EC₅₀ of 70 nM. In our experimental conditions, a simultaneous stimulation of capillaries and the arteriole with 500 nM PGE₂ led to a constriction, suggesting that the arteriolar effect prevails when both vascular segments are exposed to PGE₂. A possible explanation for these observations is that the microvascular response to PGE₂ has a kinetic component in which the fast constriction predominates over the slower capillary-mediated response. However, this stimulation via the bath perfusion likely does not reflect in vivo conditions. Accordingly, we tested the effect of increasing our ejection duration in vivo such that PGE₂ not only stimulated the targeted capillaries but also spread upstream to the arteriole. Here too this maneuver produced arteriolar constriction, again suggesting that this response will predominate when the arteriole is exposed directly to PGE₂. Since the dense capillary network within the brain lies in close proximity to all neurons (Nishimura et al., 2007; Blinder et al., 2013), it is expected that cECs are the primary sensors of neuronal activity and any neurally derived PGE₂. Therefore, the vasodilatory effect would be expected to predominate during neurovascular coupling in physiological conditions and our data suggest that this occurs through local exposure of the capillaries to PGE₂. However, a variety of brain conditions, including ischemia and neurodegeneration (Minghetti, 2004), are known to up-regulate COX-2 expression in excitatory neurons which results in an EP1-dependent neurotoxicity of PGE₂ (Kawano et al., 2006). In this pathological situation, higher PGE₂ concentrations could lead to arteriolar constriction and then contribute to PGE₂ neurotoxicity by limiting local blood supply.

Neuronal activation leads to rapid increases in blood flow within, and on the surface, of the brain. Hillman and colleagues elegantly provided evidence for involvement of the endothelium in stimulus-evoked, conducted vasodilation from the brain parenchyma to arterioles and pial arteries in vivo (Chen et al., 2014). Our previous study on neurovascular coupling demonstrated how capillary endothelium is capable of transmitting an electrical signal to cause upstream vasodilation in support of functional hyperemia (Longden et al., 2017). We showed that activation of the Kir2.1 channel in cECs by extracellular K⁺ ions propagates a regenerative hyperpolarization from cell-to-cell up to the feeding arteriole to cause vasodilation (Longden et al., 2017; Harraz et al., 2018a; MacMillan and Evans, 2018). Interestingly, recent work from Thakore et al. (2021) showed that 4-hydroxynonenal (4-HNE), an endogenous product of lipid peroxidation, activates transient receptor potential ankyrin 1 (TRPA1) channel in cECs to cause upstream arteriolar dilation during functional hyperemia. These recent findings introduce the concept that a slowly propagating short-range Ca²⁺ signal is initiated in the capillary endothelium and converted into the fast-propagating hyperpolarization that causes dilation of upstream arterioles. The conversion is proposed to occur in the transitional region between the capillaries and the arteriole (Ratelade et al., 2020) by activation of the small- and intermediate-conductance Ca²⁺-activated K⁺ channels (SK and IK, respectively) and amplification of the hyperpolarization by Kir channel (Thakore et al., 2021). The transitional region refers to the first segment sprouting out of the arteriole, visible on Figure 1 micrographs, while local stimulations are applied onto the 3rd and 4th order capillary branches after the transitional region. A vast body of literature reports (de Wit et al., 1999; Domeier and Segal, 2007; Bagher and Segal, 2011) supports the concept that a propagating Ca²⁺ signal is capable of acting through IK/SK channels, which are not present in cECs (Longden et al., 2017) but are expressed by transitional and arteriolar ECs (Hannah et al., 2011; Thakore et al., 2021). The generated hyperpolarizing signal is further amplified via Kir channel activation (Sonkusare et al., 2016) and conveyed through myoendothelial junctions to adjacent SMCs. In the dilation induced by capillary stimulation with PGE₂, inhibition of the Kir2.1 channel with Ba²⁺ had a profound effect, particularly measurable on the propagation of the dilation, which is consistent with the biphasic propagative model proposed by Thakore et al. (2021). Interestingly, inhibition of TRPA1 channels did not prevent PGE₂ from causing upstream dilation, suggesting a different initiation mechanism, likely involving G_q protein activation and inositol trisphosphate (IP₃)-mediated Ca²⁺ release, as opposed to direct Ca²⁺ entry across the plasma membrane. Our previous work also highlighted the role of G_qPCR activation in breaking down phosphatidylinositol 4,5-bisphosphate (PIP₂), resulting in decreased Kir2.1 channel activity and increased open probability of the Ca²⁺/Na⁺-permeable TRPV4 channel (Harraz et al., 2018a,b, 2020). However, blocking TRPV4 channels had no effect on the dilation induced by PGE₂ either. Given these data, we propose that EP1-initiated IP₃-dependent Ca²⁺ signals arriving in the transitional segment activate endothelial IK/SK channels, and the ensuing membrane potential hyperpolarization activates Kir channels, converting the incoming Ca²⁺ signal into a Kir-dependent hyperpolarizing signal. The characterization of such a Ca²⁺ signal will require more extensive investigation, but the vasodilation induced by capillary stimulation with PGE₂ is clearly central to the postulated role of this molecule as a neurovascular coupling agent.

Cerebral SVDs have emerged as a central link between two major co-morbidities. They account for more than 30% of strokes worldwide and at least 40% of dementia cases (Pantoni, 2010; Iadecola, 2013). CADASIL is caused by dominant mutations in the NOTCH3 receptor, expressed by SMCs and pericytes, that stereotypically lead to the extracellular deposition of the NOTCH3 ectodomain (NOTCH3^ECD), which recruits and aggregates other proteins on vessels, ultimately forming deposits termed granular osmiophilic material (GOM) (Joutel et al., 2000, 2016; Chabriat et al., 2009). One of these proteins is the tissue inhibitor of metalloproteinases 3 (TIMP3), which directly complexes with NOTCH^ECD and abnormally accumulates in the extra cellular matrix of brain vessels in patients and mice with CADASIL (Monet-Leprêtre et al., 2013). A deficit in CBF hemodynamics, including functional hyperemia, is an early disease manifestation in patients (Chabriat et al., 2000; Pfefferkorn et al., 2001; Liem et al., 2009; Huneau et al., 2018) and a prominent feature of the well-established TgNotch3^R169C CADASIL mouse model used in the present study (Joutel et al., 2010; Capone et al., 2016; Dabertrand et al., 2021). Our recent work indicates that TIMP3 effects on cerebrovascular reactivity are attributable to inhibition of ADAM17 and subsequent suppression of EGFR signaling by inhibition of ectodomain shedding of its ligand HB-EGF (Dabertrand et al., 2015, 2021; Capone et al., 2016).

Consistent with this model, we previously found that EGFR activation with exogenous soluble HB-EGF restores cerebral arterial tone and functional hyperemia (Dabertrand et al., 2015, 2021; Capone et al., 2016). Here, we found that PGE₂-induced dilation was impaired in the CADASIL mouse model and fully restored by HB-EGF.

We previously identified two downstream consequences of the suppressed TIMP3-ADAM17-EGFR signaling module: (i) the upregulation of voltage gated K⁺ (K_V1.5) channels in the arteriolar SMCs (Dabertrand et al., 2015; Capone et al., 2016); and (ii) the partial inhibition of Kir2.1 channels in cECs, but not in arteriolar ECs and SMCs (Dabertrand et al., 2021). Here, we report a third consequence: the disruption of PGE₂-induced capillary-to-arteriole signaling, reinforcing the concept that extracellular matrix alterations have profound impacts on cerebrovascular dynamics in SVDs. Using computational modeling, we previously investigated the impact of K_V channel upregulation on membrane potential dynamics in the context of concurrent activation of myocyte Kir channels (Koide et al., 2018). Interestingly, while these analyses showed that a higher K_V channel current density would reduce the membrane potential range over which Kir channels can be activated to cause and propagate dilation, the more hyperpolarized resting membrane potential (9 mV) actually facilitates Kir channel activation. Thus, arterioles from the CADASIL mouse model would still hyperpolarize and dilate in response to Kir channel opening, as observed experimentally (Dabertrand et al., 2015; Koide et al., 2018), but at the cost of a smaller vasodilatory reserve. At the capillary level, we previously described, and modeled, how a 50% reduction in cECs Kir2.1 current is sufficient to completely abolish the capillary-to-arteriole electrical signaling in response to 10 mM K⁺ (Harraz et al., 2018a; Moshkforoush et al., 2020), and then strongly reduces functional hyperemia in the CADASIL mouse model (Dabertrand et al., 2021). We attributed this endothelial dysfunction to a reduced cEC metabolism caused by inhibition of the EGFR pathway (Dabertrand et al., 2021). The resulting lower ATP/ADP ratio in CADASIL compared to WT cECs decreases the synthesis of PIP₂ and its availability to act as an essential cofactor for Kir2.1 channel—hence reducing the channel activity (Huang et al., 1998; Hansen et al., 2011; Harraz et al., 2018a, 2020). Of particular note, PIP₂ is also a substrate for phospholipase C during EP1 signaling, and a reduced pool of PIP₂ would certainly limit the synthesis of the second messenger IP₃, which mobilizes Ca²⁺ from endoplasmic reticulum stores through its action on its cognate receptor. The observation that HB-EGF restores both Kir2.1- and PGE₂-initiated signaling is consistent with this concept. The impact of CADASIL on neurovascular coupling is thus multifaceted, involving disruption of two different propagating signals sharing connections to the EGFR pathway in cECs, and Kir channel activation in the transitional and arteriolar segments.

We have made major progress in establishing potential contributing mechanisms to cerebral hemodynamics impairment observed at an early stage of CADASIL, a Mendelian paradigm of SVDs (Chabriat et al., 2009) and the most common hereditary cause of stroke (Pantoni, 2010) and dementia (Schmidt et al., 2012). Here we demonstrate that functional hyperemia deficits in this SVD involve a PGE₂-initiated capillary-to-arteriole signal that is normally regulated by the TIMP3-ADAM17-EGFR signaling module. Furthermore, the evidence that PGE₂ induces arteriolar dilation via capillary stimulation has the potential to reconcile disparate findings in neurovascular studies. Our mechanistic studies thus lay the groundwork for novel targeted strategies for treating CADASIL and other cerebrovascular diseases.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado, Anschutz Medical Campus.

AR, JF, and FD performed ex vivo experiments, data collection, and analysis. TL and NW performed in vivo experiments, data collection, and analysis. AJ contributed to the study design. FD designed and directed the research and wrote the manuscript. All authors edited the manuscript and approved its submission.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GW declared a shared affiliation, with no collaboration, with one of the authors AJ to the handling editor at the time of the review.

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