Shear stress induced KLF4 is a key determinant in AVM pathogenesis

Background Vascular networks form, remodel and mature under the influence of both fluid shear stress (FSS) and soluble factors. For example, FSS synergizes with Bone Morphogenic Protein 9 (BMP9) and BMP10 to promote vascular stability. Mutation of the BMP receptors ALK1 and Endoglin or the downstream effector SMAD4 leads to Hereditary Hemorrhagic Telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). But how endothelial cells (ECs) integrate FSS and BMP signals in normal vascular development and homeostasis, and how mutations give rise to malformations is not well understood. Results Here we show that loss of Smad4 in the murine ECs leads in the one hand to increased sensitivity to flow and the resulting AVMs are characterized by excessive elongation and polarity against the flow and in the other hand, blocks the anti-proliferative effects of high FSS. Cellularly, we identified increased proliferation-mediated loss of arterial identity as the main event triggering AVM formation in Smad4 depleted ECs. Molecularly, we found that flow-induced excessive KLF4-PI3K/AKT-CDK6 pathway activation mediates the enhancement in morphological responses to flow triggering AVMs. Conclusions Our study showed that loss of polarization against the flow is not required for AVM formation in SMAD4 ECko. Instead, increased EC proliferation and loss of arterial identity due to PI3K/Akt-Cdk6 hyperactivation and Klf4 over-expression are the main events associated with AVM formation.


Introduction
Vascular networks form, remodel and mature under the influence of multiple mechanical and biochemical signals, but how these are integrated to promote vascular development and adult homeostasis is not well understood. Fluid shear stress (FSS) from blood flow is a critical variable that determines vascular endothelial cell (EC) number, shape and movement in vascular development and maintenance 1 . One aspect of EC flow responses is the existence of a cellautonomous shear stress set-point specific to each vessel type. FSS near the set-point promotes EC elongation and alignment parallel to the flow, and stabilizes the vessel whereas flow that is persistently above or below this level triggers vessel remodeling to restore FSS to the appropriate magnitude 2 . ECs also polarize and migrate according to the flow direction; in different systems this may be with or against the flow 3 , but in the developing retina is against the flow, which is proposed to be important in guiding vessel formation 4 .
We previously found that shear stress within the physiological range synergizes with secreted Bone Morphogenic Protein (BMP) 9 and BMP10 to activate Smad 1/5, which promotes EC quiescence and vascular homeostasis. This pathway contributes to the inhibition of EC proliferation by FSS and to expression of factors that mediate pericyte recruitment, thus, stabilizing the vessels 5  These findings suggest that the canonical BMP9/10-Smad signaling plays a crucial role in shear stress regulation of vascular homeostasis and that AVMs arise from disruption of these mechanisms. One important mediator downstream of this pathway is PI3K/AKT, which is hyperactivated in HHT lesions in human patients and in mouse models 8,12,13 . Pharmacological inhibition of PI3K or depletion of EC Akt1 rescued AVM formation in HHT murine models 8,13 .

Smad4 signaling maintains the shear stress set-point-mediated EC responses
Impaired responses of EC to FSS including migration direction, proliferation and changes in EC size and cell fate have proposed to mediate HHT lesions, mainly in models of HHT1 and HHT2, ie., mutations in ENG and ALK1 7,15 . To explore flow-mediated EC events in JP-HHT, where SMAD4 is mutated, we depleted primary human umbilical cord ECs (HUVECs) for SMAD4 using small interfering RNA (siRNA) vs CTRL siRNA (confirmed in Figure 1G). Cells were subject to laminar shear stress at 1 or 12 DYNES/cm 2 for 24 and 48 hours (Figure 1A-I To test these observations in vivo, we measured the ratio length/width of individual EC labelled for VE cadherin and ERG within the capillaries in Cdh5-Cre negative (Smad4 Fl/Fl-control) versus AVMs in tamoxifen inducible Smad4 EC specific deficient postnatal day 6 (P6) (Smad4 iΔEC ) retinas. These measurements confirm increased morphological EC responses to flow upon Smad4 depletion ( Figure 1J,K,L). Together, these results imply that SMAD4 signaling restricts shear stress-mediated EC shape responses to flow.
ECs in the postnatal retina polarize and migrate against the flow direction, from the veins towards the arteries, with the degree of polarization correlating with shear stress magnitude 4 .
Disrupted polarization and impaired movement of ECs against the direction of flow has been proposed to mediate AVM formation in Eng and Alk1 mutants 7,15 . We therefore analyzed polarity in capillaries and AVMs in Smad4 Fl/Fl and Smad4 iΔEC P6 retinas by staining for Golph4 to label the Golgi apparatus, Erg for the EC nuclei and Isolectin B4 (IB4) to visualize the endothelium ( Figure 1M,N). The relative position of the Golgi and nuclei were then quantified ( Figure 1O).
EC polarization against the predicted flow direction was moderately increased in Smad4 iΔEC ( Figure 1M,N,O). Thus, multiple EC morphological responses to shear stress are increased after SMAD4 KO in vitro or Smad4 ECko in vivo.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint It well established that physiological high FSS inhibits EC proliferation 16 . As expected 8 , labelling of Smad4 Fl/Fl and Smad4 iΔEC retinas for the mitotic marker KI67 and the total EC marker IB4 revealed increased EC proliferation in AVMs (Figure 1Q,P,R). In vitro, EdU labeling to identify cells in S phase showed that SMAD4 depletion increased baseline cell cycle progression and completely blocked the inhibition by high shear stress ( Figure 1S). Thus, Smad4 is required for flow-mediated repression of EC proliferation. Taken together, these results show that Smad4 resembles Alk1 and Eng in that it is also required for flow-induced quiescence but is opposite in that it suppresses rather than enhances morphological responses to flow.

Smad4 depletion induced PI3K/AKT activation regulates flow-mediated EC responses
We previously identified an increased in PI3K/AKT activity upon inactivation of BMP9/10-Alk1-Smad4 in ECs, which was further augmented by high FSS 8,13 . To further understand if increased responsiveness of SMAD4 deficient cells to FSS is due to PI3K/AKT pathway activation, we subjected CTRL siRNA versus SMAD4 depleted HUVECs to increasing magnitudes of shear stress (1-5-12 DYNES/cm 2 ). SMAD4 deletion significantly increased AKT phosphorylation at serine 473, a marker of activation, under static condition and increasing flow magnitudes had an additive effect (Figure 2A,B). To assess the role of activated AKT in the amplified response to FSS, we inhibited PI3K/AKT signaling for 48 hours using a specific PI3K inhibitor-Pictilisib To test in vivo, we treated Smad4 Fl/Fl and Smad4 iΔEC pups with Pictilisib and examined retinas labeled with IB4, Erg and Golph4. Here, we found that PI3K inhibition blunted the axial polarity in both Smad4 Fl/Fl and Smad4 iΔEC retinas (Figure 2 I,J,K). EdU labeling in these mice showed that inhibition of AKT rescued the excessive EC proliferation in Smad4 iΔEC vascular plexus ECs ( Figure 2L). In vitro, inhibition of PI3K also reversed the excess cell cycle progression after SMAD4 depletion in ECs under high FSS ( Figure 2M). Thus, endothelial SMAD4 functions to restrain flow-induced PI3K/AKT and downstream responses including elongation, polarization against the flow and proliferation but not the EC alingment.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint

High flow-induced KLF4 is a key determinant in AVM formation
Within the retinal developing plexus, the shear stress levels are the highest in the vascular plexus close to the optic nerve, and gradually decreasing toward the sprouting front 17,18 . To gain insights into Smad4 regulation in this context, we labelled Smad4 Fl/Fl and Smad4 iΔEC retinas for Krüppel like transcription factor (Klf4), a gene that shows strong, dose-dependent induction by FSS 19 , and for IB4 ( Figure 3A-D'). In control, Smad4 Fl/Fl retinas, Klf4 expression was minimal in the low shear vascular front and capillary ECs (Figure 3A), moderate in higher flow large veins, increased further in larger arteries, and at the highest intensity at the first retinal branch points where the wall shear stress is maximal (arrows in Figure 3B,B'). This specific region corresponds to the location where the AVMs most often form 8 . In Smad4 iΔEC retinas, Klf4 expression was highly upregulated in AVMs at the highest intensity relative to the feeding artery and vein (arrows in Figure 3D  Previous studies have shown that high expression of Klf2, a close homolog of Klf4 induces EC elongation 21 . We therefore considered its role in the altered behaviours of Smad4 ECko or knockdown cells. To test its function in vivo, we generated two genetic models. First, we examined EC specific Tx-inducible Klf4 LOF neonates (Klf4 i∆EC ) where AVMs were induced by administration of blocking antibodies (blAb) for BMP9/10 ( Figure 3G,H) and secondly, we created EC specific Tx-inducible double ko mice, Smad4;Klf4 i∆EC ( Figure 3I,J). Tx was injected at P1-P3 and retinas were analysed at P6. Efficient Smad4 and Klf4 gene deletion was validated by qPCR from P6 mouse lung endothelial cells (mLECs; Figure 3K was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint ( Figure 3H,J,L,M). Thus, shear stress-induced Klf4 is a key determinant in AVM pathogenesis and the first molecular marker identified to-date to discern flow-dependent AVM formation from flow-independent excessive sprouting.

KLF4 mediates flow-induced hyper-responsiveness of SMAD4 depleted HUVECs
To address mechanisms, we next investigated which of the aberrant EC flow responses require KLF4. HUVECs depleted for either SMAD4 or KLF4 or both, SMAD4;KLF4 using the siRNA strategy were grown without flow and with low or high flow for 48 hours (Figure 4A Figure 4N). Klf4 OE thus induces many of the key effects of Smad4 deletion. Together, these results show that Klf4 contributes to the morphological effects mediated by Smad4 LOF.

KLF4 mediates the shear stress induced aberrant behaviours in AVMs
To address EC Golgi orientation, Fl/Fl, Smad4 iΔEC , Klf4 iΔEC and Smad4;Klf4 iΔEC retinas were examined. Compared to Fl/Fl mice, Klf4 deficient capillaries showed reduced polarization against the flow direction; in Smad4 iΔEC retinas, additional Klf4 inactivation blunted the increased axial polarity (Figure 5A-C). Klf4 ko ECs were less elongated than Fl/Fl ECs and Klf4 inactivation rescued the excessive elongation of Smad4 iΔEC ECs, thus confirming our in vitro findings ( Figure 5D). To assay Klf4-mediated EC cell cycle progression, we injected EdU into Tx induced non-CDH5 Fl/Fl, Smad4 iΔEC and Smad4;Klf4 iΔEC P6 pups, 4 hours before labeling the retinas for EdU and ERG ( Figure 5E). As previously observed, ERG+/EdU+ double positive ECs increased markedly in Smad4 iΔEC retinas, exclusively in AVMs. Klf4 inactivation significantly decreased the number of ERG/+EdU+ in the vascular plexus of Smad4 iΔEC retinas to comparable . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint levels to Fl/Fl retinas (Figure 5E,F). Elevated Klf4 thus contributes to increased polarity and proliferation in Smad4 ECko blood vessels.

Flow induced KLF4 acts upstream of mechanosensory complex-PI3K/AKT pathway
To untangle the relationships between KLF4 and PI3K, we inhibited PI3K/AKT with Pictilisib and subjected HUVECs to 5 DYNES/cm 2 for 2 hours. RT-PCR results show no effect of Pictilisib on KLF4 under any of these conditions ( Figure 6A). We also considered the role of the junctional mechanosensory receptor complex that mediates flow responses including PI3K activation 22,23 .
Depletion of each of the components of the mechanosensory receptor complex had no effect on the flow upregulation of KLF4 expression ( Figure 6B). Thus, KLF4 expression does not require PI3K or the mechanosensory junctional receptor complex.
We then tested effects of KLF4 on AKT activation, with and without flow and SMAD4 knockdown. KLF4 inactivation led to a decrease in pAKT in static conditions, blunted the increase in Akt activity under flow and rescued AKT hyperactivation in SMAD4 depleted HUVECs, in both static and flow conditions ( Figure 6C,D). To further test if flow-induced KLF4 is upstream of PI3K we examined the KLF4 OE HUVECs. KLF4 upregulation was sufficient to activate AKT, with or without FSS (Figure 6E,F). To test these findings in vivo, we labelled retinas for phosphorylated S6 ribosomal protein (pS6), a downstream target of AKT activation, as well as IB4. Smad4 iΔEC retinas showed high pS6 as expected, which was largely rescued by Klf4 deficiency (Figure 6G-I). KLF4 is thus upstream of PI3K to control EC elongation and proliferation after Smad4 ECko.

Increased EC proliferation triggers AVM formation in Smad4 deficient AVMs
Current models propose that decreased polarization and migration of ECs against the direction of flow is critical in AVM formation upon Eng and Alk1 ECko. The current data show that AVM formation upon Smad4 depletion involves, if anything, improved polarity. These findings prompted us to investigate if increased EC proliferation is the main event driving AVMs.
Performing cell cycle distribution in ECs FACS sorted from Smad4 Fl/Fl versus Smad4 iΔEC retinas, we revealed an increase in actively cycling ECs in S/G2/M together with a decrease in ECs in G1, confirming increased EC proliferation upon Smad4 depletion ( Figure 7A).
Cell cycle progression is tightly regulated by members of the cyclin dependent kinase (CDK) family. To identify dysregulated cell cycle regulators upon SMAD4 LOF, we performed WB . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint analysis to multiple cell cycle regulators. We observed increased phosphorylation of Retinoblastoma (RB) and expression of E2F1 transcription factor after Smad4 depletion, together with an elevated CDK2 and CDK6 protein levels ( Figure 7B). KLF4 inactivation alone led to reduced levels in all the main cell cycle regulators, suggesting cell cycle arrest. KLF4 inactivation in SMAD4 LOF HUVECs normalized levels of pRB1, E2F1, CDK2 and CDK6 ( Figure 7B).
To test whether effects on cell cycle are the main drivers of AVMs, we treated Smad4 Fl/Fl and Smad4 iΔEC pups with Palbociclib, a specific inhibitor of CDK4/6 shown to efficiently inactivate RB and block cell cycle progression 24 ( Figure 7C). As expected, labeling retinas for KI67 and IB4 showed decreased EC proliferation ( Figure 7D) and significant rescue of AVMs ( Figure   7E). Taken together, these results suggest that increased cell proliferation as a result of flowinduced excessive KLF4-AKT-CDK4/6 drives AVM formation.
Recent data show that cell cycle arrest is a prerequisite for maintaining arterial identity 24 .
To further assess the connection between cell cycle and arterial identity, we analyzed HUVECs depleted for KLF4 and AKT, or treated with Palbociclib to inhibit cell cycle progression. RT-PCR identified a significant increase in expression of the arterial markers EPHRINB2 and SOX17 upon inhibition of KLF4-AKT-CDK4/6 pathway (Figure 7F,G).
To test these results in vivo, we labeled retinas for Sox17 ( Figure 7H). In Fl/Fl retinas, Sox17 was confined to ECs in main arteries and a few arterioli. In AVMs in Smad4-deficient retinas, Sox17 expression was completely abrogated. In Klf4 ECko retinas, Sox17 expression expanded towards the vein and capillary ECs. Klf4 inactivation in Smad4 iΔEC retinas largely rescued Sox17 expression in arteries. CDK4/6 inhibition led to even greater expansion of Sox17 expression in capillary and venous ECs and restored Sox17 expression in Smad4 ECko arteries ( Figure 7H). Collectively, these results suggest that increased EC proliferation-mediated loss of arterial identity is the main cell event triggering AVM formation.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made           was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint

Discussion
We have revealed here that loss of Smad4 in mouse ECs triggers increased sensitivity to FSS with elevated PI3K/Akt signaling and Klf4 expression, enhanced elongation and polarization in FSS simultaneolsy with inefficient FSS-mediated cell cycle blockade and loss of arterial identity due to increased CDK2/CDK6 protein levels. It has been proposed that blood flow is 'a second hit' contributing to HHT, as murine AVMs develop in regions of high shear stress 5,8 , but the mechanisms by which shear stress contributes to AVM pathology remains largely undefined. ECs display an intrinsic set-point for shear stress that determines signaling and gene expression outputs.
Previous work showed that VEGFR3 expression is one factor that can determine shear stress setpoint for different types of vessels 2 . Non-canonical WNT signaling was proposed to modulate axial polarity set-point to control vessel regression in low flow regions 26 . Our results identify SMAD4 signaling as a novel mechanism that "sets the set-point" for high flow-mediated EC quiescence responses: elongation, alignment and orientation. Smad4 is also critical for FSS mediated growth suppression and arterial EC fate, yet, it remains to be determined if these cell events are also linked to changes in the set point.
We previously identified an increased in PI3K/AKT signaling to be responsible for AVM formation 8,13 . Herein, we provide genetical evidence that flow-induced Klf4 further augments this pathway and contributes to AVM pathogenesis. However, the mechanism by which Klf4 regulates Akt activation requires further investigation. Interestingly, identification of this mechanism for high flow AVMs resembles molecularly other vascular malformations. The low flow venous malformations are due to increased PI3K/AKT by GOF mutations in PI3K and therefore activation of AKT 27 , and similarly, GOF mutations in PI3K act as a second genetic hit in cavernous malformations, where MEKK3 activated Klf4 is the primary mechanism 28 .
It is generally assumed that the mechanisms of AVM formation are similar if not identical, in HHT1, HHT2 and JP-HHT. Our data, however, argue that AVMs are not strictly identical, at least in mouse models. We found that SMAD4 depleted cells elongate in the absence of flow and elongate further and align in response to much lower FSS. These changes presumably reflect effects on the cytoskeleton and/or cell junctions, which would be consistent with elevated PI3K 21 , but remain to be defined for Klf4. In Eng and Alk1 LOF mice, failure to migrate against the flow . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint was the main event proposed to trigger AVM formation 29,30 . Opposingly, our data suggest more migration against the flow in the AVMs upon Smad4 ECko. Nevertheless, AVMs are characterized by gain of venous markers and contain exclusively venous like ECs. In line with a recent report emphasizing a venous origin for AVMs upon Smad4 LOF, our observed effects on EC polarity might thus explain the enhanced migration from veins towards the arteries giving rise to AVMs 31 .
These differences in flow driven migration in different mouse models may indicate either distinct cellular and molecular mechanism driving AVM formation in different mouse models, or that axial polarity/reverse migration is not important in AVM formation. As there is yet no functional evidence that migration against the flow is important in Alk1 and Eng LOF AMVs, based on our findings, we propose instead that loss of flow-induced suppression of EC proliferation is the key cellular event triggering AVM formation upon Smad4 depletion.

Arterial specification induced by HSS requires cell cycle arrest, a process in which Notch through
Cx37-p27signaling is a key element 24 . Our results suggest that loss of Smad4 leads to loss of arterial identity due to an increased in flow-induced excessive KLF4-PI3K/AKT-CDK6 mediated EC proliferation.
In summary, our study identified Smad4 signaling as a novel mechanism to maintain EC quiesncence by restraining flow-induced KLF4-PI3K/AKT-CDK6 activation in ECs. The mechanism by which Smad4 "sets the set-point" for FSS-mediated cel responses to maintain EC quiescence and what are the subsequent molecular events implicated, is an important direction for future studies. AVMs in HHT patients form later in life. Although our study was conducted in a developmental setting, this mechanism is likely to be important for maitenance of vascular homeostasis also n mature vessels. Thus Klf4-PI3K/Akt-CDK6 may be an attractive target for HHT patients treatment.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. The PI3K inhibitor Pictilisib (Selleckchem, S1065, 20 mg/kg/day) and CDK4/6 inhibitor Palbociclib (Selleckchem, S1116, 70 mg/kg/day) were administered intraperitoneally (i.p) at P4 and P5.
Mice were maintained under standard specific pathogen-free conditions, and animal procedures were approved by the animal welfare commission of the Regierungspräsidium Karlsruhe (Karlsruhe, Germany). . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Quantitative real-time PCR
RNAs from HUVECs or mouse lung ECs (mLECs) were purified using RNeasy-kit (74106, Qiagen  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint were co-transfected with lentiviral vector and packaging plasmids (pCMV-dR8.91 and pCMV-VSV-G) using X-treme GENE 9 reagent (Sigma). Culture supernatant containing viral particles was collected 36 and 72 h after transfection and concentrated by centrifugation at 1500g for 60 min at 4℃. The pellets were resuspended in 1 mL of PBS and stored at -80℃. For virus infection, Huvecs cells were transduced with optimal volume of lentiviral virus at 50% confluence in MV2 medium and 8μg/ml Polybrene (Sigma). After 24 h, the medium containing viral particles was replaced with fresh medium and after additional 24h, the infected cells were selected with 2 μg/ml puromycin for 48 h.

Exposure of endothelial cells to increased shear stress
HUVECs transfected with siRNAs or OE-HUVECs were plated in a six-well plate and on an orbital shaker (Rotamax120, Heidolph Instruments) at 50, 150 or 250 rpm to generate laminar sheer stress of 1, 5 or 12 DYNES/cm 2 respectively. Results were confirmed in a µ-Slide VI 0.4 (Ibidi, 80601) using a pump system (Ibidi, 10902).

Western blotting
HUVECs were washed with PBS and lysed with Laemmli buffer (1610740, Biorad). Samples were separated on 10% SDS-PAGE gels and transferred on 0.2µm nitrocellulose membranes (10600004, GE Healthcare). Western blots were developed with the Clarity Western ECL Substrate (1705061, Biorad) on a Luminescent image Analyzer, Fusion FX (Vilber). Bands' intensity were quantified using ImageJ.

Proliferation Assay
Proliferation analysis was performed using Click-iT EdU Alexa Fluor 488 Imaging kit (Life Technologies). P6 pups were injected with 200 μg of EdU (5 mg/mL) and sacrificed 4 hours later.
EdU staining was done according the manufacturer's protocol.

Statistical analysis
All data are shown as mean ± standard error of the mean (SEM). Samples with equal variances were tested using Mann-Whitney U test or two-tailed Student's t-test between groups. P value <0.05 was considered to be statistically significant. Statistical analyses were performed for all quantitative data using Prism 9.0 (Graph Pad).
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 4, 2022. ; https://doi.org/10.1101/2022.07.04.498236 doi: bioRxiv preprint