Introduction

Vascular smooth muscle cells (VSMCs) are contractile mural cells wrapping around endothelial cells (ECs) of large vessels, especially arteries (Ando et al., 2022; Donadon & Santoro, 2021; Stratman et al., 2020). The expression of contractile proteins, such as alpha-smooth muscle actin (encoded by acta2), distinguishes VSMCs from pericytes, the other type of mural cells primarily associated with small vessels (Bahrami & Childs, 2020; Donadon & Santoro, 2021; Stratman et al., 2020). VSMCs are important for arterial homeostasis (Basatemur, Jorgensen, Clarke, Bennett, & Mallat, 2019). In the brain, VSMC constriction and relaxation is essential for functional hyperemia in neurovascular coupling (Hill et al., 2015; Kaplan, Chow, & Gu, 2020). Phenotype switching or dedifferentiation of VSMCs, marked by lower expression of contractile proteins, is prominent in pathological progression of atherosclerosis (Basatemur et al., 2019). VSMC phenotype switching or dedifferentiation also contributes to cerebrovascular diseases and neurodegeneration (Aguilar-Pineda et al., 2021; Chou et al., 2022; Frosen & Joutel, 2018; Milewicz et al., 2010; Oka et al., 2020; Poittevin et al., 2014). As development and regeneration share many common molecular and cellular mechanisms (Nowotarski & Sánchez Alvarado, 2016), insights on VSMC differentiation on brain arteries during development may inform strategies to reprogram and regenerate dedifferentiated VSMCs and alleviate cerebrovascular diseases.

VSMCs differentiate from heterogenous perivascular progenitors of mesoderm and neural crest origins (Ando et al., 2019; Donadon & Santoro, 2021; Whitesell et al., 2019). Previous research described various mechanisms that regulate VSMC differentiation. Autonomous Notch activation is required for specification of pdgfrb+ mural cell progenitors from peri-arterial mesenchyme, and these progenitors later differentiate into acta2+ VSMCs (Ando et al., 2019). Arterial Notch signaling activated by blood flow is necessary for acta2+ VSMC emergence on zebrafish trunk dorsal aorta (X. Chen, Gays, Milia, & Santoro, 2017). Chemokine signaling promotes VSMC association with zebrafish dorsal aorta, whereas blood flow responsive transcription factor krüppel-like factor 2 (encoded by klf2a in zebrafish) prevents their association with the adjacent cardinal vein (Stratman et al., 2020). Endothelial BMP signaling and autonomous foxc1 expression regulate VSMC differentiation in zebrafish ventral head (Watterston, Zeng, Onabadejo, & Childs, 2019; Whitesell et al., 2019). Autonomous expression of an ATP-sensitive potassium channel modulates VSMC differentiation from pdgfrb+ progenitors in the brain (Ando et al., 2022). These studies suggest that VSMC differentiation is highly organotypic.

The circle of Willis (CoW) consists of major arteries that supply blood to the vertebrate brain, including internal carotid arteries (ICAs) and posterior communicating arteries (PCAs) (Campbell et al., 2019; Schröder, Moser, & Huggenberger, 2020). In early development, ICAs are the first large arteries to form and supply all circulation in the developing brain (Kathuria, Gregg, Chen, & Gandhi, 2011; Menshawi, Mohr, & Gutierrez, 2015). In adults, posterior circulation becomes independent, and ICAs remain major feeding arteries in anterior, while connected to posterior circulation with PCAs, completing a circle of arteries (Kathuria et al., 2011; Schröder et al., 2020). In adults, intracranial aneurysms are frequently found close to CoW arteries (Frosen & Joutel, 2018). CoW arteries are wrapped by VSMCs, and VSMC dedifferentiation and hyperplasia are described in carotid atherosclerosis and Moyamoya disease (Chou et al., 2022; Fox, Dorschel, Lawton, & Wanebo, 2021). Spatiotemporal dynamics of VSMC differentiation on vertebrate CoW arteries during development have not been thoroughly investigated. VSMC differentiation is difficult to observe in vivo with mammalian models, as mammalian embryos develop in utero and depend on maternal circulation. Zebrafish is an important vertebrate model for vascular development, as zebrafish embryos are externally fertilized, optically clear, and thus accessible to confocal live imaging of developing blood vessels (Stratman & Weinstein, 2021). Establishment of zebrafish fluorescent transgenic reporter lines with pdgfrb and acta2 promoters enables in vivo visualization and assessment of mural cells and VSMCs (Ando et al., 2016; Whitesell et al., 2014). In addition, zebrafish CoW shows comparable geometry to human CoW: the caudal division of the internal carotid artery (CaDI) in zebrafish is comparable to human ICA, and the posterior communicating segment (PCS) in zebrafish is comparable to human PCA (Isogai, Horiguchi, & Weinstein, 2001). Taking advantage of the zebrafish model, we describe the spatiotemporal pattern of VSMC differentiation on CoW arteries, and associate this pattern with blood flow activation of klf2a in arterial ECs.

Results

Artery muscularization is spatiotemporally regulated in CoW arteries

In zebrafish brain, arteries are formed by ECs from cranial venous plexus (Fujita et al., 2011). Recent single-cell transcriptome profiling of vascular cells in prenatal human brain also supports an endothelial differentiation trajectory from proliferative venous ECs to quiescent arterial ECs (Crouch et al., 2022). To establish the temporal sequence of CoW arterial specification and VSMC differentiation, we started with confocal live imaging of fluorescent transgenic reporter lines Tg(flt4:yfp)^hu4881, which labels primitive venous ECs, and Tg(kdrl:hras-mcherry)^s896, which is expressed in all ECs but enriched in arterial ECs, at four stages starting at 32 hours post fertilization (hpf), when CoW arteries are assembled (Chi et al., 2008; Hogan et al., 2009; Isogai et al., 2001). As arterial ECs express higher kdrl, we used the intensity of mcherry as an indicator of artery specification (Figure 1A-1E) (Chi et al., 2008). We found that when ECs from cranial venous plexus migrated to form CoW arteries at 32 hpf, they still retained primitive venous identity, as indicated by flt4 expression (Figure 1A). When connections between cranial venous plexus and CoW arteries disappeared at 54 hpf, CoW arteries no longer expressed primitive venous flt4 (Figure 1B). Expression of arterial enriched kdrl in CaDI and PCS at 3 days post fertilization (dpf) was significantly higher than 32 hpf (Figure 1C and 1E). At 4 dpf, kdrl expression in CoW arteries remained comparable to 3 dpf, suggesting that arterial specification of CoW was completed by 3 dpf (Figure 1C-1E). Together, these data suggest CoW morphogenesis precedes arterial endothelial gene expression.

Figure 1.

Previous research suggested that acta2+ VSMCs on CoW arteries differentiate from pdgfrb+ mural cell progenitors (Ando et al., 2022; Ando et al., 2019). Thus, to characterize spatiotemporal dynamics of VSMC differentiation on CoW arteries, we performed confocal live imaging of Tg(acta2:mcherry)^ca8, TgBAC(pdgfrb:egfp)^ncv22, and Tg(kdrl:cerulean)^sd24, to visualize respectively VSMCs (red), pdgfrb+ progenitors (green), and developing arteries (white) at four stages starting from 32 hpf (Figure 2) (Ando et al., 2016; Page et al., 2013; Whitesell et al., 2014). We found that pdgfrb+ progenitors emerged on CoW arteries around 54 hpf after CoW morphogenesis, and most pdgfrb+ progenitors did not express acta2 at 54 hpf (Figure 2A-2B, 2E-2F, and S1A-S1B). The number of pdgfrb+ progenitors on CoW arteries increased temporally from 54 hpf to 3 dpf (Figure 2B, 2C, and 2E). Notably, while many pdgfrb+ cells on CaDI differentiated into acta2+ VSMCs by 3 dpf, acta2+ VSMCs were hardly observed on adjacent BCA or PCS, suggesting that VSMC differentiation started between 54 hpf and 3 dpf on CaDI but not BCA or PCS (Figure 2B-2C, 2E-2F, and S1A-S1B). At 4 dpf, the number of pdgfrb+ cells on CoW arteries remained comparable to 3 dpf, while the number of acta2+ VSMCs on CaDI increased (Figure 2C-2F and S1A-S1B). Importantly, most pdgfrb+ cells on BCA and PCS only started to express acta2 around 4 dpf, much later than CaDI (Figure 2C-2F and S1A-S1B). These findings support that differentiation of acta2+ VSMCs from pdgfrb+ progenitors progresses spatially and temporally along the anterior to posterior direction in CoW arteries.

Figure 2.

CoW arteries have spatiotemporal difference in hemodynamics

Our data suggest that VSMCs on CaDI of anterior CoW differentiate much earlier than BCA and PCS of posterior CoW, although all acta2+ VSMCs on CoW differentiated from pdgfrb+ progenitors after they were recruited to CoW (Figure 2 and S1). In addition, anterior and posterior CoW vascular morphogenesis and arterial specification occurred around the same time (Figure 1). We speculate that hemodynamic distribution may contribute to the spatiotemporal difference in VSMC differentiation on CoW arteries. CaDI receive proximal arterial feed through lateral dorsal aorta from cardiac outflow tract (Isogai et al., 2001). Furthermore, CaDI differs from the BCA or PCS in terms of overall vascular geometry, which is among determinants of local vascular hemodynamic forces such as wall shear stress (WSS) (Katritsis et al., 2007). To explore this possibility, we performed microangiography with a high molecular weight fluorescent dextran to determine each CoW artery diameter. We found that there was no significant different among CaDI, BCA and PCS (Figure 3A-3D). Then, we measure red blood cell (RBC) velocity by axial line scanning microscopy to analyze Tg(kdrl:gfp;gata1:DsRed)^zn1/sd2 zebrafish at 54 hpf, 3 and 4 dpf. The RBC velocity across CoW arteries was differentially distributed, with CaDI having the highest RBC velocity from 54 hpf to 4 dpf (Figure 3E-3G). This is consistent with CaDI being the inlet vessel for arterial blood flow within the entire CoW circulation. Next, we used computational fluid dynamics (CFD) to simulate the effect of flow on CoW, based on the Dextran-filled CoW geometric network and RBC velocity (ANSYS, 2014; Z. Chen et al., 2019; Fernandes et al., 2022). We found an overall increase in average WSS from 54 hpf to 4 dpf (Figure 3A-3C, and 3H). Further, the simulated flow pattern and WSS on the CoW showed that WSS in CaDI was significantly higher than BCA and PCS at 54 hpf, 3 dpf, and 4 dpf (Figure 3A-3C, 3I-3K). Moreover, upon comparing WSS with the number of acta2+ VSMCs per CoW arteries during the development, we observed a Pearson correlation coefficient of r =0.595, indicating a moderate to strong positive correlation between these two variables (Figure S1C). In summary, the analysis of blood flow suggests that CoW arteries undergo varied hemodynamic WSS, which correlates with the spatial and temporal pattern of VSMC differentiation along CoW arteries.

Figure 3.

Blood flow is required for CoW artery muscularization

Our data suggest that ECs under arterial flow might favor acta2+ VSMC differentiation from pdgfrb+ progenitors. To test this hypothesis, we set up an in vitro cell co-culture experiment where GFP-PDGFRB+ human brain pericytes (GFP-HBVPs) were cultured in flow amenable outlet slides, then covered by a thin layer of collagen type 1. Confluent human endothelial cells (HUVECs) were seeded on top of that and then exposed to steady-state laminar or pulsatile flow conditions (Figure 4A) (Abello, Raghavan, Yien, & Stratman, 2022). Differentiation of PDGFRB+ human brain pericytes into ACTA2+ VSMCs was then quantified after 24 hours of flow treatment. We found that brain pericytes under pulsatile flow were larger and showed higher expression of VSMC differentiation markers including PDGFRB, ACTA2, and TRANSGELIN (Robin et al., 2013) (Figure 4B-4D), suggesting that pulsatile flow favored PDGFRB+ progenitor differentiation into VSMC.

Figure 4.

Previous research suggests that blood flow is dispensable for recruitment of pdgfrb+ progenitors on BCA and PCS (Ando et al., 2016). To corroborate this, we injected 0.35 ng of tnnt2a morpholino antisense oligonucleotide (MO) into Tg(pdgfrb:egfp, kdrl:hras-mcherry)^ncv22/s896 at one to two-cell stage, to abrogate cardiac contractility and thus blood flow (Ando et al., 2016; Chi et al., 2008; Sehnert et al., 2002). Indeed, the number of pdgfrb+ cells on CaDI, BCA, and PCS at 3 dpf in tnnt2a morphants was comparable to uninjected sibling control, suggesting blood flow did not affect pdgfrb+ progenitor recruitment to CoW arteries (Figure S2G-S2H). To determine the effect of blood flow on VSMC differentiation, we injected tnnt2a MO into Tg(acta2:mcherry, kdrl:gfp)^ca8/zn1 (Cross, Cook, Lin, Chen, & Rubinstein, 2003; Sehnert et al., 2002; Whitesell et al., 2014). Notably, we found no acta2+ VSMCs on CoW arteries of tnnt2a morphants at either 3 or 4 dpf (Figure 4E-4F and 4H-4I), suggesting that blood flow was required for acta2+ VSMC differentiation on CoW arteries.

To further explore the temporal requirement of blood flow, we treated embryos with 25 μM nifedipine, a drug shown to reduce heart rate in zebrafish embryos (Figure S2A-S2C) (Gierten et al., 2020), from 54 hpf, right before the onset of pdgfrb+ progenitors’ expression of acta2 (Figure 2A-2C). We found that at 3 dpf the number of acta2+ VSMCs on the CaDI of treated embryos is greatly reduced (Figure 4G and 4J); acta2+ VSMCs on BCA and PCS at 4 dpf are also reduced significantly (Figure 4G and 4K), suggesting that blood flow reduction after 54 hpf impaired VSMC differentiation on CoW arteries.

To determine whether blood flow is required for short-term VSMC maintenance after differentiation on CoW arteries, we treated embryos with nifedipine from 4 dpf, after VSMC differentiation on CoW arteries, and imaged at 5 dpf. The number of acta2+ VSMCs on the CaDI and PCS in treated embryos was comparable to untreated controls (Figure S2D-S2F), suggesting that blood flow was required for differentiation but not for short-term maintenance of CoW VSMCs.

Blood flow-regulated transcription factor klf2a modulates CoW artery muscularization

Our results suggest that CoW ECs might express a flow dependent transcriptional program that regulates spatiotemporal dynamics of VSMC differentiation on CoW arteries. Previous studies showed that expression pattern of transcription factor Klf2 in arterial ECs closely follow the pattern of flow induced WSS (Lee et al., 2006; Parmar et al., 2006). Consistently, higher Klf2 expression can be induced by unidirectional pulsatile flow (Dekker et al., 2002). Furthermore, klf2 is implicated in VSMC migration on arterial blood vessels during mouse development: loss of klf2 leads to aorta VSMC deficiency (Wu, Bohanan, Neumann, & Lingrel, 2008). Interestingly, klf2a expression in primitive veins of zebrafish trunk prevents VSMC association (Stratman et al., 2020). Hence, we tested the hypothesis that klf2a might be spatiotemporally regulated in CoW ECs and modulate VSMC differentiation. We first imaged Tg(klf2a:h2b-egfp, kdrl:hras-mcherry)^ig11/s896, which labels nuclei with active klf2a signaling, and quantified the number of klf2a+ ECs in CoW arteries (Chi et al., 2008; Steed et al., 2016). To account for overall changes in EC number during development (Ulrich, Ma, Baker, & Torres-Vazquez, 2011), and length of each vessel, we also imaged Tg(fli1:nls-gfp, kdrl:hras-mcherry)^y7/s896, which labels all EC nuclei (Figure S3A-S3I) (Roman et al., 2002). From 32 hpf to 54 hpf, the number of klf2a+ EC in CaDI was comparable to PCS (Figure 5A-5C), however, the number of klf2a+ ECs in CaDI was significantly higher than BCA and PCS from 3 dpf (Figure 5A and 5D-5E), when acta2+ VSMCs differentiated on CaDI but not BCA and PCS (Figure 2B-2C, 2F, and S1A-S1C). Pearson correlation comparing the number of klf2a+ ECs with the number of acta2+ VSMCs in CaDI, BCA and PCS at 3 dpf showed strong positive correlation with r = 0.974 (Figure S5J). These results suggest that the number of klf2a+ ECs in CoW arteries is spatially correlated with VSMC differentiation.

Figure 5.

To further define the role of klf2a in VSMC differentiation on CoW arteries, we knocked down klf2a in Tg(acta2:mcherry, kdrl:gfp)^ca8/zn1 with 11 ng MO injection at one to two-cell stage and imaged at 3 to 4 dpf (Nicoli et al., 2010; Whitesell et al., 2014; Zhong et al., 2006). We validated the effect of klf2a MO knockdown with Tg(klf2a:h2b-egfp, kdrl:hras-mcherry)^ig11/s896 (Figure S3G-S3I) (Chi et al., 2008; Steed et al., 2016). Compared to uninjected control, klf2a morphants have significantly less acta2+ VSMCs on CaDI at 3 dpf but a normal number by 4 dpf (Figure 6A-6D), suggesting that klf2a knockdown delayed VSMC differentiation on CaDI. Together these data suggest that endothelial klf2a is required for the onset of VSMC differentiation on CoW arteries.

Figure 6.

Discussion

In this study, we employed confocal live imaging of fluorescence transgenic zebrafish embryos to investigate the spatiotemporal dynamics of vascular smooth muscle cell (VSMC) differentiation on the cerebral vasculature (CoW), which comprises major arteries supplying blood to the vertebrate brain. Our observations revealed that CoW morphogenesis precedes the expression of arterial endothelial genes. Specifically, mural cell progenitors marked by pdgfrb+ initiate the expression of the VSMC marker acta2 subsequent to their recruitment to CoW arteries. Notably, VSMC differentiation occurs earlier in the anterior CoW arteries compared to their posterior counterparts, owing to the elevated wall shear stress (WSS) resulting from a higher velocity of red blood cells in the incoming blood flow. To investigate the regulatory role of blood flow on the spatiotemporal dynamics of VSMC differentiation, we employed an in vitro co-culture assay along with genetic manipulation and drug treatments. Our findings indicate that blood flow indeed governs the timing and location of VSMC differentiation on CoW arteries. Moreover, we observed that the flow-responsive transcription factor klf2a is activated in a gradient manner from anterior to posterior CoW arteries, preceding the onset of VSMC differentiation. Knockdown experiments targeting klf2a revealed a delay in VSMC differentiation specifically in the anterior CoW arteries. Taken together, these findings highlight the role of endothelial klf2a activation by blood flow as a mechanism that promotes VSMC differentiation on CoW arteries within the brain (see Figure 7 for a visual summary).

Figure 7.

Previous research shows that in zebrafish trunk, as soon as recruited to the dorsal aorta, VSMCs express acta2 and transgelin (tagln), suggesting simultaneous recruitment and differentiation from sclerotome progenitors (Ando et al., 2016; Stratman et al., 2017). On CoW arteries in zebrafish brain, however, VSMC differentiation occurs after pdgfrb+ progenitor recruitment and proceeds from anterior to posterior (Figure 2B-2F). The observed spatiotemporal dynamics of CoW VSMC differentiation again highlights organotypic development of blood vessels.

Our data suggests klf2a mediate blood flow regulation of VSMC differentiation on brain arteries, and thus raises the question of how klf2a transduces endothelial signals to mural cell progenitors and VSMCs. Notch signaling appears a plausible downstream effector of klf2a activation, as previous research suggests Notch responds to flow in heart valve development (Fontana et al., 2020), possibly downstream of klf2 (Duchemin, Vignes, & Vermot, 2019). Wnt signaling is another possible downstream effector of klf2a, as previous research suggests endocardial klf2 upregulates Wnt signaling in neighboring mesenchymal cells in heart valve development (Goddard et al., 2017). The roles of Notch and Wnt signaling in organotypic VSMC differentiation on brain arteries remain to be determined.

The expression of klf2a in CoW arterial ECs remained stable from 3 dpf to 4 dpf (Figure 5), which raises and interesting question on whether sustained klf2a expression supports further maturation of VSMCs, as acta2+ VSMCs on CaDI showed distinct morphology at 4 dpf compared with 3 dpf, when they started differentiation from pdgfrb+ mural cell progenitors (Figure 2C-2D and S1). Previous research found that acta2+ VSMCs on the BCA and PCS express pericyte enriched abcc9 (ATP binding cassette subfamily C member 9) at 4 dpf (Ando et al., 2022), when they started differentiation from pdgfrb+ mural cell progenitors and expressing acta2; abcc9 expression is gradually lost from 5 dpf to 6 dpf (Ando et al., 2022). In addition, acta2+ VSMCs on CaDI, BCA, and PCS still retain expression of pdgfrb at 6 dpf (Ando et al., 2021). Thus, it is possible that stable expression of klf2a in CoW arterial ECs supports further VSMC maturation, as indicated by expression of tagln (Colijn, Nambara, & Stratman, 2023). Another aspect of VSMC maturation is its function to confer vascular tone. A previous study found that VSMC covered vessels in zebrafish brain dilate as early as 4 dpf and constrict at 6 dpf (Bahrami & Childs, 2020). Future study may focus on the association between expression of different VSMC markers and VSMC functional maturation.

Another question is how flow activates klf2a in brain arteries. In a hypoxia-induced pulmonary hypertension model, klf2 is activated by G-protein coupled receptor mediated Apelin signaling (Chandra et al., 2011). In heart valve development, endocardial klf2a is upregulated by membrane bound mechanosensitive channels trpp2 and trpv4 (Heckel et al., 2015). The pathway through which flow activates klf2a in brain arterial ECs remains unknown. The CoW VSMC phenotype of klf2a morphants is similar to, but do not fully recapitulate the phenotypes of tnnt2a morphants or nifedipine treated embryos (Figure 4E-4L and Figure 6). A proximal explanation is compensation by paralogous klf2b in zebrafish. Further characterization of CoW VSMC development in klf2a and klf2b genetic mutants (Rasouli et al., 2018; Steed et al., 2016) may help determine whether klf2b compensates klf2a in CoW VSMC differentiation.

In addition to WSS, transmural pressure and associated mechanical stretch is another mechanical input of vascular wall that may contribute to VSMC differentiation. VSMCs autonomously sense and adapt to mechanical stretch (Haga, Li, & Chien, 2007). Cyclic stretching activates arterial VSMC production of certain components of extracellular matrix (ECM) (Leung, Glagov, & Mathews, 1976). In turn, specific interactions between ECM and integrins enable VSMCs to sense mechanical stretch (Goldschmidt, McLeod, & Taylor, 2001; Wilson, Sudhir, & Ives, 1995). Connection to ECM is essential for VSMC force generation (Milewicz et al., 2017). VSMCs express stretch activated trpp2, which enables myogenic response to autoregulate resting arterial diameter (Sharif-Naeini et al., 2009). Interestingly, ex vivo stretching promotes expression of contractile proteins, such as acta2 and tagln in VSMCs on murine portal veins (Albinsson, Nordstrom, & Hellstrand, 2004; Turczynska, Hellstrand, Sward, & Albinsson, 2013). In addition, excessive mechanical stretch promotes VSMC dedifferentiation and inflammation (Cao et al., 2017; Wang et al., 2018). Further investigation is needed to determine the potential role of increasing transmural pressure and associated mechanical stretch during development in VSMC differentiation.

There are a few limitations to our current study. Genetic manipulation and drug treatment that reduce heart rate would also reduce nutrients carried by blood flow, and the effects of nutrient and flow reduction could not be uncoupled in live zebrafish embryos. In addition, these methods are only capable of qualitative reduction of flow but not specific dampening of pulsations. In vitro three-dimensional (3D) vascular culture models, which combine ECs and mural cells (Mirabella et al., 2017; Vila Cuenca et al., 2021), could be further optimized to simulate complex geometry of brain arteries. Combining these models with microfluidics, which allows precise calibration of nutrient composition in culture media, flow velocity and pulse, would enable more thorough analysis of endothelial mechanotransduction and its contribution to VSMC differentiation (Abello, Raghavan, Yien, & Stratman, 2022; Gray & Stroka, 2017; Griffith et al., 2020).

We used nifedipine to temporally reduce heart rate and blood flow. Nifedipine is a blocker of L-type voltage-dependent calcium channels (VDCC) (Quevedo et al., 1998). A previous study shows that ML218, a T-type VDCC-selective inhibitor, tends to increase VSMC differentiation (Ando et al., 2022). In addition to the difference in drug targets, we also noted that in the previous study, the increase in VSMC differentiation only occur on anterior metencephalic central arteries (AMCtAs) that are more than 40 μm away from the BCA; these AMCtAs are much smaller than CoW arteries and have different geometry (Ando et al., 2022). Although the most obvious effect of nifedipine in zebrafish embryos is heart rate and blood flow reduction (Gierten et al., 2020), it is possible that nifedipine affect VSMC through VDCCs. Further investigation is needed to uncouple the roles of different VDCCs in VSMC differentiation and their association with hemodynamics.

We used Tg(flt4:yfp, kdrl:ras-mcherry)^hu4881/s896 to show CoW arterial specification from primitive venous ECs (Chi et al., 2008; Hogan et al., 2009). kdrl is not the best arterial marker as its expression is only enriched but not restricted to arteries. While the use of the two fluorescence transgenic lines helped us establish the temporal sequence of CoW morphogenesis, arterial specification and VSMC differentiation, analysis of additional arterial and venous markers is needed to fully characterize arterial specification during vertebrate brain vascular development.

In summary, our work identifies flow induced endothelial klf2a activation as a mechanism that regulates spatiotemporal dynamics of VSMC differentiation on CoW arteries in the vertebrate brain. Our data may help advance approaches to regenerate differentiated VSMCs in cerebrovascular diseases. It would be important to further investigate mechanisms upstream and downstream of klf2a activation in brain arterial ECs and additional mechanotransduction pathways that may contribute to VSMC differentiation on brain arteries.

Material and Methods

Zebrafish husbandry and transgenic lines

Zebrafish were raised and maintained at 28.5 °C using standard methods. Protocols are approved by the Yale Institutional Animal Care and Use Committee (2020-11473). TgBAC(pdgfrb:egfp)^ncv22 was generated by Ando et al., has been reported that the pdgfrb:egfp+ cells express other pericyte enriched marker abcc9 (ATP binding cassette subfamily C member 9) (Ando et al., 2016; Ando et al., 2019). By this transgenic line, they also observed that pdgfrb mutant lack brain pericytes and exhibit loss of vascular smooth muscle coverage (Ando et al., 2021). Thus, we used TgBAC(pdgfrb:egfp)^ncv22 to study mural cell coverage of vascular endothelium in zebrafish CoW arteries. All transgenic lines in Table 1 were established previously.

Table 1.

Confocal fluorescence microscopy

Zebrafish embryos were raised in 0.003% 1-phenyl-2-thiourea (PTU, phenylthiocarbamide, or n-phenylthiourea, Sigma P7629) from gastrulation stage to prevent pigmentation. Embryos imaged live by confocal fluorescence microscopy were anesthetized in 0.1% tricaine methanesulfonate (TMS, MS-222, or Syncaine, Western Chemical, NC0872873) and mounted in 1% low melt agarose within glass bottom microwell dishes. Fluorescence images were captured with an upright Zeiss LSM 980 confocal microscope using a 20X objective.

Image analysis

Confocal fluorescence images were analyzed with Imaris microscopy image analysis software (Bitplane, Oxford Instruments). Average fluorescence intensity of mcherry in Tg(flt4:yfp, kdrl:hras-mcherry)^hu4881/s896 was estimated by generating volume objects covering the artery of interest with Surface module. Vessel lengths of the arteries were manually traced with Filament module. Numbers of pdgfrb+ mural cell progenitors, acta2+ vascular smooth muscle cells, and klf2a+ and fli1+ endothelial nucleus were counted with Spots module.

Morpholino injections

Morpholino antisense oligonucleotides were synthesized by Gene Tools. Morpholinos in Table 2 were validated previously. Optimized dose injected into each embryo are listed. Uninjected siblings were used as controls.

Table 2.

Nifedipine treatment

Nifedipine was dissolved into 20 mM in dimethyl sulfoxide (DMSO). The stock solution was diluted into 25 μM in egg water with 0.003% PTU to treat zebrafish embryos from 54 hpf to 3 dpf or 4 dpf. The stock solution was diluted into 20 μM in egg water with 0.003% PTU to treat zebrafish embryos from 4 dpf to 5 dpf. The same volume of DMSO was added into egg water with 0.003% PTU for sibling control embryos.

Microangiography

Embryos were anesthetized in 0.1% tricaine methanesulfonate, placed on an agarose mold, and injected pericardially with 4 nL Tetramethylrhodamine Dextran (2,000,000 molecular weight, Thermo Fisher) at a concentration of 10 mg/mL. Subsequently, the vasculature of embryos was checked for Dextran fluorescence signal under a stereomicroscope (Olympus, MVX10). Embryos with Dextran fluorescence were mounted in 1% low melt agarose within glass bottom microwell dishes and imaged with confocal microscope.

Red blood cell velocity

Red blood cell (RBC) velocity was measured by used axial line scanning to analyze zebrafish in the Tg(kdrl:gfp;gata1:DsRed)^zn1/sd2 background, in which blood vessels and RBCs are labeled with green and red fluorescent protein, respectively (Cross et al., 2003; Traver et al., 2003). As described previously (Barak et al., 2021), this axial line scan was conducted in a Zeiss LSM980 confocal microscope. A line was drawn inside the lumen of CoW vessels and parallel to the longitudinal axis of the vessel. The average length of the line was 21.21 μm. The scan speed was chosen as maximum per second for 20,000 cycles (total scan duration was 5.86 s; line scan time is 292.80 μs). A kymograph was exported in TIFF format after every measurement by ZEN 3.5 and analyzed RBC recognition and segmentation by using Fiji (ImageJ). Firstly, the RBC kymography image was processed using ‘noise despeckle’ and ‘enhance contrast’ steps. The brightness and contrast were appropriately adjusted if needed. Next, the RBCs were segmented, thresholding was adjusted, and the slope of the RBCs was calculated automatically using the ‘Analyze particle’ option. Finally, the velocity of each RBC was calculated using the following equation: velocity = (1/tan(a)) * (length of the y axis of the kymograph/duration of the total scan). We averaged systolic peak velocities and end diastolic velocities to measure the mean RBC velocity in the CoW vessels.

Computational fluid dynamic (CFD) simulation

Computational fluid dynamic (CFD) simulation was performed according to the geometry model of the circle of Willis (CoW) at 54 hpf, 3 and 4 dpf as previously described with modification (ANSYS, 2014; Barak et al., 2021). First, three-dimensional (3D) geometry models of CoW arteries were reconstructed from confocal z-stack microangiography images of Dextran injected Tg(kdrl:gfp) embryos with the Filament module in Imaris (9.9.0, Bitplane, Oxford instruments). These CoW 3D geometry models were pre-processed in ANSYS SpaceClaim 2022 R1 software. For example, the computational mesh on the CoW vascular wall was created and the boundary conditions were set up for the simulation. Meshing orthogonal quality was calculated, and objects less than 0.01 were excluded. Inlets and outlets were determined in the 3D model for the flow simulation. Next, the pre-processed models were imported in ANSYS Fluent 2022 R1 software. The non-Newtonian behavior of blood flow was modeled using the Carreau model which was provided by ANSYS. Blood density was considered as 1,060 kg/m³. The computational simulation used average RBC velocity among the CoW arteries at 54 hpf (532.72 μm/s), 3 dpf (602.34 μm/s) and 4 dpf (552.83 μm/s), respectively. After the CoW geometry and numerical solution of blood flow were ready, the flow simulation was implemented with 200 iterations to identify the magnitude and distribution of the WSS in the CoW.

Cell Culture

Pooled primary Human Umbilical Vein Endothelial Cells (HUVEC) (PCS-100-010^TM, ATCC) were seeded at an initial concertation of 5,000 cells per cm², in 1x M119 media (110343-023, Gibco) supplemented with 16% FBS (Gibco), 84 µg/mL of heparin sodium salt (H3393, Sigma-Aldrich), 25 µg/mL of endothelial cell growth supplement (02-102, EMD Millipore Corp.) and 1x Antibiotic-antimycotic solution (15240-062, Gibco). Cell cultures were maintained at 37°C, 5% CO₂ and 95% humidity, until the cell reached 80% confluence.

To facilitate cell visualization in cell co-cultures, Human Brain Vascular Pericytes (HBVP) (1200, ScienCell) were transfected using lentiviral particles (LVP310, GenTarget, Inc.) to induce GFP expression under EF1a promotor. Cells cultures were initiated by seeding 5,000 cells per cm² in 175 cm² plastic flasks pre-coated with gelatin and 1x DMEM (11995-065, Gibco) supplemented with 10% FBS (Gibco), and 1x Antibiotic-antimycotic solution under the same cell culture conditions described above. When cell cultures were at 80% confluence, 200 µL containing 2 x 10⁶ GFP-lentiviral particles were added to each 175 flasks. After 72 hours, fresh cell culture media supplemented with 10µg/mL of Blasticidin (15205, Sigma-Aldrich) were added to each flask to select the positive transfected cells.

Flow assays and immunostaining

HBVP cells were harvested and seeded at a concentration of 1.3 x 10⁵ cells in 0.4 optical plastic flow microslides (80176, Ibidi) precoated with 1 mg/mL gelatin and incubated for 24 hours under standard culture conditions. After the initial incubation, 100 µg/mL of collagen I (354249, Corning) diluted in DMEM cell culture media was added to the slides to create a thin layer on top of the HBVP cells. After 2 hours, the media was removed and 2.5 x 10⁵ HUVECs were seeded on top of the collagen I layer and incubated for additional 24 hours. After cell co-cultures were established, the slides were exposed to laminar (15 dyn/cm²) or pulsatile (12-15 dyn/cm²) flow for 24 hours, implementing a peristaltic pump adapted to produce different types of flow (Abello, Raghavan, Yien, & Stratman, 2022). After 24 hours, cultures were rinsed with 1x PBS and fixed for 30 minutes in 4% paraformaldehyde at room temperature. Cell cultures were immunostained with α-Smooth muscle actin D4K9N XP^® rabbit monoclonal antibody (19245, Cell Signaling), followed by Alexa Fluor 633 goat anti-rabbit IgG (A21071, Invitrogen). Confocal images were obtained using a 40x objective with a W1 Spinning Disk confocal microscope, a Fusion camera, and the Nikon Eclipse Ti2-E base. Fiji image processing software was used for image analysis and fluorescence intensity quantification.

Statistics

All statistical analyses were performed with GraphPad Prism (version 10.0.3). Mann–Whitney test was used to compare two groups to test mean differences (protein level, morpholino and nifedipine treatment). Two-way ANOVA followed by Tukey’s multiple comparisons was used to test mean differences compared more than two groups under different conditions (average fluorescence intensity, cell number, vascular diameter). Ordinary one-way ANOVA with Tukey’s multiple comparisons was used to test mean differences among three groups (RBC velocity, WSS, and cell number). Pearson correlation analysis was used to compare the strength and direction of a linear relationship between two variables with a covariance (WSS vs acta2+ EC, acta2+ EC vs klf2+ EC).

Acknowledgements

We thank the labs of S. Schulte-Merker, D.Y.R. Stainier, S.J. Childs, N. Mochizuki, D. Traver, J. Vermot, B.M Weinstein, and L.I. Zon for sharing zebrafish transgenic lines. We thank N. Semanchik for all the assistance with zebrafish adult colonies and husbandry. Experiments in the manuscript were supported by R01NS109160 and R01DK118728 awarded to S.N. and AHA23POST1025829 post-doctoral fellowship awarded to I.F.X. The paper is based on a dissertation submitted by S.C. to fulfill in part the requirements for the degree of Doctor of Philosophy, Yale University. S.C. was supported by a Gruber Science Fellowship from Yale Graduate School of Arts and Sciences.

Author Contributions

S.C. and I.F.X contributed to the experiments, the data analysis, and composed the figures. R.W. performed dextran microangiography experiments. J.A. and A.N.S. performed the in vitro experiments. S.C., I.F.X and S.N. wrote the manuscript and prepared figures. J.A. and A.N.S. edited the paper.

Supplementary Figures and Supplementary Figure Legends

Figure S1.

Figure S2.

Figure S3.