Loss of Gucy1a3 Causes Poor Post-Stroke Recovery by Reducing Angiogenesis Via the HIF-1α/VEGFA Signaling Pathway in Mice

Man Luo Guangxi Medical University First A liated Hospital Dongcan Mo Guangxi Medical University First A liated Hospital LiuYu Liu Guangxi Medical University First A liated Hospital Jianli Li Guangxi Medical University First A liated Hospital Jing Lin First A liated Hospital of Nanchang University Jie Liang Sun Yat-sen University First A liated Hospital Fei Ye Sun Yat-sen University First A liated Hospital Xiaoju Wu Guangxi Medical University First A liated Hospital Xiaoling Li Guangxi Medical University First A liated Hospital Jiaoxing Li Sun Yat-sen University First A liated Hospital Wenli Sheng (  shengwl@mail.sysu.edu.cn ) Sun Yat-sen University First A liated Hospital https://orcid.org/0000-0001-6867-371X


Introduction
Ischemic stroke is one of the leading causes of death and disability worldwide [1] . Although tissue plasminogen activator and endovascular thrombectomy have been established as effective therapies for ischemic stroke [2] , only a small population of patients bene ts from these treatments because of the short therapeutic time window. Hence, there is an urgent need for effective therapeutic approaches, and the development of effective therapeutic strategies that can improve the recovery of blood ow and functional outcomes after stroke is highly desirable.
Angiogenesis refers to the formation of new blood vessels from pre-existing vasculature [3] . It occurs in the ischemic penumbra within hours after stroke and lasts for weeks. Cumulative evidence has indicated that post-ischemic angiogenesis is crucial for increasing the blood supply in the penumbral areas and improving the prognosis of stroke patients [4][5][6] . Therefore, developing new therapies to augment angiogenesis is of utmost importance for the treatment of ischemic stroke.
The Gucy1a3 gene encodes the α1 subunit of soluble guanylate cyclase (sGC), which is the most abundant sGC isoform in the vascular system. sGC is a heterodimeric enzyme composed of one α and one β subunit. The β subunit also associates with the α2 subunit, which is expressed at a lower level. sGC is the major receptor for nitric oxide (NO). NO binds the heme iron of sGC to induce cyclic guanosine monophosphate (cGMP) production, which then activates the cGMP-dependent protein kinase pathway [7] . It is widely accepted that NO plays an important role in angiogenesis [8,9] . Previous studies have reported that administration of an NO donor to rats after stroke signi cantly enlarged vascular perimeters and increased the numbers of proliferating cerebral endothelial cells and newly generated vessels in the ischemic boundary regions [10] . Mice lacking endothelial nitric oxide synthase showed severe impairment of angiogenesis in response to limb ischemia, and administration of an NO donor accelerated angiogenesis [11] . In addition, both in vitro and in vivo experiments have demonstrated that NO can promote endothelial cell proliferation and migration and tip cell sprouting, all of which are important steps in angiogenesis [12][13][14] .
Different mutations or polymorphisms in the Gucy1a3 gene are associated with various vascular phenotypes, including ischemic stroke [15] , moyamoya cerebral arteriopathy [16] , coronary artery disease [17] , and pulmonary artery hypertension [18] . A recent study found that homozygous Gucy1a3 risk allele (rs7692387) carriers were at increased risk of ischemic events early after coronary stenting [19] . In addition, a previous study reported that sGC α1 subunit de ciency in a mouse model of transient middle cerebral artery occlusion (MCAO) signi cantly reduced cerebral blood ow, enlarged infarct volumes, and aggravated neurological de cits, suggesting that the sGC α1 subunit is protective in ischemic stroke [20] . However, whether suppression of GUCY1A3 function can inhibit vascular regeneration in the brain after stroke is unknown.
In this study, we aimed to examine the role of GUCY1A3 in the regulation of angiogenesis in the ischemic brain. A Gucy1a3 knockout (Gucy1a3-KO) mouse model was generated to investigate the effects and potential molecular mechanisms of GUCY1A3 in cerebral ischemia-induced angiogenesis.

Animals
Male C57BL/6J mice (8-to 10-week-old, 21-25g) were used. All animal experiments were performed in accordance with approved protocols of the Sun Yat-sen University Animal Care and Use Committee and were in line with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health in China. Mice were maintained on a 12-hour light/dark cycle at 22 ± 2°C and allowed access to food and water ad libitum before the experiments. Gucy1a3 knockout (KO) mouse model was established by the CRISPR/Cas9 system, which was described in detail in the Supplementary Materials.

Permanent Middle Cerebral Artery Occlusion (pMCAO) for Stroke Model
The left middle cerebral artery was permanently occluded using the method described previously [21] .
Brie y, mice were anaesthetized with chloral hydrate (200mg/kg, intraperitoneal injection), and their fur and skin were disinfected. The left common carotid artery, bifurcation of the internal common carotid artery (ICA), and external common carotid artery (ECA) were carefully dissected. A 5-0 nylon lament, with its tip rounded by heating near a ame, was inserted through the ECA stump and gently advanced (about 10 mm) into the left internal carotid artery until it blocked the origin of the MCA. The wound and skin were was closed and the lament left in place. The body temperature of the mice was maintained 37.0 ± 0.5°C with a heating pad during surgery.

Neurological Scoring
Neurological de cit scores were evaluated at 24 h and 96h after pMCAO as follows [22] : 0, no de cit; 1, exion of contralateral forelimb upon lifting the whole animal by the tail; 2, circling to the contralateral side; 3, falling to the contralateral side; and 4, no spontaneous motor activity.

Infarct volume
The cerebral infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The brains of mouse with 96 hours after pMCAO surgery were taken directly after anesthesia, and they were frozen quickly in -20°C refrigerator for about 20 min. Then the brain was taken out and placed on a special brain trough for 5 consecutive slices with a thickness of 2mm. The slices were placed in 2% TTC (Sigma Aldrich, MO, USA), soaked for 30min at 37℃ with shaded light, and nally xed with 4% paraformaldehyde. After the above staining, red represents normal brain tissue, and white represents infarcted brain tissue. The sections were arranged in sequence, labeled, scaled and photographed, and the volume of cerebral infarction was calculated by ImageJ analyzer software (National Institutes of Health, USA). The infarct area of each slice was calculated, and then the total infarct volume was calculated by multiplying the infarct area of each slice by the thickness (2mm).

Immuno uorescence
Ninety-six hours after the pMCAO procedure, the brains were collected quickly and post-xed in 4% paraformaldehyde overnight. Serial coronal sections of the mouse brain (8µm thick) were cut using a cryostat. Sections were incubated with 3% BSA for 30 min at 37°C and then immune-stained with primary antibodies against CD31 (endothelial cell marker, 1:1000, Abcam, MA, USA) and VEGFA antibody (1:250, Abcam, MA, USA). Following overnight incubation, the sections were washed and subsequently incubated with secondary antibodies Cy3-conjugated donkey anti-mouse IgG and Alexa 488 IgG (Cys for red and Alexa 488 for green, 1:1000, Invitrogen, CA, USA) for 1 h at 37°C. Images obtained using a uorescence microscope (Leica-LCS-SP8-STED, Germany). And images area and intensity were measured in ImageJ.

Microvascular density (MVD)
CD31 was used to mark endothelial cell and evaluate the MVD as previously described [23] . Brie y, the CD31 + cells apart from the vessel, no matter alone or in groups, would be regarded as a microvessel and should be counted. 6 slices of a group were counted and the number of microvessels was counted in ve sights of 400× eld (i.e., 40×objective lens and 10×ocular lens) for calculating the MVD (number of microvessels per mm 2 ). Counting was done by two independent observers and the results are expressed as means ± SD.

Western Blot Analysis
The brain tissue from the peri-infarct region was collected at 96hours after stroke. Tissue samples were homogenized in RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing PMSF (Beyotime Biotechnology, Shanghai, China) and crushed by an ultrasonic comminution machine. After a 30min centrifugation at 12000 rpm, the supernatant (the total protein) was harvested. After that, the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the proteins were transferred onto the PVDF membranes. Subsequent to blocking with QuickBlock blocking buffer for 30 min at 25˚C followed by three washes for 10 min with western wash buffer, the membranes were incubated overnight at 4˚C with SGC α1 antibodies (1:3000, Abcam, MA, USA), HIF1α mouse monoclonal antibody (1:3000, A nity, OH, USA), Vegfa monoclonal antibody (1:2000, Invitrogen, CA, USA) and GAPDH antibody (1:5000, A nity, OH, USA). Following three washes for 10 min with TBST, the samples were incubated with the secondary antibody, goat anti-rabbit immunoglobulin G (1:3000, Abcam, MA, USA) and membrane was nally visualized using the chemiluminescence detection system (Thermo Scienti c, IL, USA).

Statistics
Statistical analyses were performed using SPSS software (SPSS Inc., IL, USA). The data were given as mean ± standard deviation. Comparisons between two groups were analyzed by Student's t tests. Data comparisons between multiple groups were analyzed by one-way ANOVAs with Tukey's post hoc tests. P values <0.05 were considered statistically signi cant.

Generation of Gucy1a3-KO mice
Gucy1a3-modi ed mice were generated via co-microinjection of Gucy1a3 single-guide RNA (targeting exon 5, Fig. 1a) and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Gucy1a3-modi ed mice were mated with wild-type (WT) mice to obtain mutant mice heterozygous for the Gucy1a3 mutation. Then, Gucy1a3 heterozygous mutant mice were intercrossed to produce homozygous Gucy1a3 global knockout mice. Homozygous Gucy1a3-KO mice were born at a normal Mendelian ratio and did not show apparent alterations or a reduced life expectancy. All mice were genotyped by sequencing for the CRISPR/Cas9induced mutation in the exon 5 of the Gucy1a3 locus, and a 13-base pair deletion was found in Gucy1a3-KO mice (Fig. 1c). Western blot analysis was performed to con rm the loss of the sGC α1 subunit. As shown in Fig. 1d, 1e, the protein was completely absent in the brains of Gucy1a3-KO mice, which con rmed at the protein level that Gucy1a3-KO mice were successfully generated.

Loss of Gucy1a3 increases the infarction volume and neurological de cits after ischemic stroke
To evaluate the protective effects of GUCY1A3 against stroke, we measured the infarct volumes and neurological de cit scores in a mouse model of pMCAO. The infarct volumes were evaluated by TTC staining 96 hours after the pMCAO operation, and the neurological de cit scores were evaluated at 24 and 96 hours after successful modeling using the Longa score (0-4 points) method. The infarct volume 96 hours after pMCAO, represented as the percentage of hemispheric volume, was signi cantly larger in Gucy1a3-KO mice than in WT mice (23.8±6.1% vs. 17.0±5.1%, P<0.05, Fig. 2a, 2b). Furthermore, there was no signi cant difference in neurological de cit scores between WT and Gucy1a3-KO mice 24 hours after pMCAO, but neurological de cits were signi cantly greater in Gucy1a3-KO mice at 96 hours after the operation (P<0.05, Fig. 2c).

Loss of Gucy1a3 impairs angiogenesis after ischemic stroke
Angiogenesis is a prerequisite for recovery from ischemic stroke; hence, we determined whether GUCY1A3 could promote angiogenesis after experimental stroke. Microvessels were assessed by calculating the areas of cells that expressed the endothelial marker CD31 in the perifocal striatum region. CD31 immuno uorescence analysis showed a much higher microvessel density in WT mice than in Gucy1a3-KO mice at 96 hours post-MCAO (Fig. 3a, 3b). This result suggests that Gucy1a3 deletion impaired angiogenesis after ischemic stroke; therefore, Gucy1a3 might promote angiogenesis after focal ischemia.

Loss of Gucy1a3 decreases angiogenic factor expression in ischemic stroke
Angiogenesis is largely driven by vascular endothelial growth factor A (VEGFA) production in response to hypoxia. To verify angiogenic dysfunction in Gucy1a3-KO mice, we detected VEGFA expression in the perifocal striatum of Gucy1a3-KO mice using immuno uorescence and western blotting. Double immuno uorescence staining of CD31 and VEGFA showed that VEGFA was primarily localized in endothelial cells. The immune expression of VEGFA was decreased in the infarcted striatum of Gucy1a3-KO mice compared with that of WT mice (Fig. 4a, 4b). Furthermore, the VEGFA protein level of ischemic mice at 96 hours post-pMCAO was signi cantly lower in the Gucy1a3-KO group, as measured by western blotting (Fig. 4c, 4e).
Hypoxia inducible factor 1α (HIF-1α) is a key regulator of VEGFA production in ischemic tissues. Inhibition of the HIF-1α signaling blocks angiogenesis in various diseases. To assess the effects of Gucy1a3-KO on HIF-1α, we analyzed the HIF-1α expression levels in the ipsilateral striatum homogenate obtained from the KO and WT groups at 96 hours post-pMCAO using western blotting. The results showed that Gucy1a3-KO decreased the HIF-1α protein level signi cantly (Fig. 4c, 4d).

Discussion
Ischemic stroke is a common clinical disease that is associated with high levels of disability and mortality and seriously threatens human life and health. In recent years, Gucy1a3, which encodes the α1 subunit of sGC, has been proposed to be tightly correlated with cerebrovascular diseases and coronary artery disease [15][16][17]24] . However, only a few studies have examined the effects of GUCY1A3 on ischemic stroke, and the mechanisms underlying these effects remain unclear. In the present study, Gucy1a3-KO mice were used to investigate the effects of GUCY1A3 on ischemic stroke. Our results indicated that loss of Gucy1a3 increased the infarct volume and aggravated neurological de cits after pMCAO. Moreover, Gucy1a3-KO brains exhibited signi cantly lower microvessel densities than WT brains at 96 hours post-pMCAO, suggesting that Gucy1a3 deletion suppressed angiogenesis after ischemic stroke. Further analysis con rmed that loss of Gucy1a3 decreased VEGFA and HIF-1α expression levels, which may contribute to the decrease in angiogenesis in Gucy1a3-KO mice after pMCAO.
The impact of genetic variations in the Gucy1a3 gene on ischemic cardio-cerebrovascular diseases has been highlighted by previous studies. Variants of the Gucy1a3 gene are enriched in patients suffering from ischemic stroke and coronary artery disease [15,24,25] . A previous study has also shown that the infarct size was larger and the neurologic outcome was worse in sGCα1 −/− mice than in WT mice subjected to 1 hour of MCAO and 23 hours of reperfusion, but the infarct volumes and neurological de cits were similar after 24 hours of permanent occlusion in both genotypes [20] . That study further revealed that impaired vascular relaxation may underlie the more marked reperfusion de cit observed in sGCα1 −/− mice than in WT mice after middle cerebral artery reperfusion [20] . Interestingly, our results showed no signi cant difference in neurological de cit scores between WT and Gucy1a3-KO mice 24 hours after pMCAO, but neurological de cits and the infarct volume were signi cantly greater in Gucy1a3-KO mice at 96 hours after pMCAO. However, temporal changes in the effects of sGCα1 de ciency on infarct volume and neurological de cits following pMCAO cannot be fully explained by impaired vascular relaxation.
Previous studies have con rmed that angiogenesis is correlated with improved functional recovery and prolonged survival after ischemic stroke [6,26] . Krupinski et al. [6] reported that angiogenic activity occurred in ischemic stroke patients 3-4 days after stroke. In rodent stroke models, endothelial cells in the periinfarct brain tissue start to proliferate as early as 12-24 hours following ischemic stroke; accordingly, vessel density signi cantly increases in the peri-infarct region 3 days after ischemia [27,28] . In our study, after 4 days (96 hours) of pMCAO, the microvessel density was lower in Gucy1a3-KO mice than in WT mice. A larger infarct volume and worse functional outcome were also observed, which might have resulted from the decreased angiogenic activity in the peri-infarct region.
VEGFA is the most important angiogenesis-promoting factor and plays an important role in angiogenesis after focal cerebral ischemia [29] . As a key oxygen concentration-dependent transcription factor, HIF-1α can regulate multiple target genes, including VEGFA, and thereby modulate angiogenesis after ischemia [30] . As mentioned previously, sGC is the major cytosolic receptor for NO, catalyzing the conversion of guanosine-5'-triphosphate to cGMP. NO and hypoxia were reported to upregulate the VEGFA gene by enhancing HIF-1 activity [31,32] . We further examined whether the decreased angiogenesis in the Gucy1a3-KO ischemic brain could be explained by the decrease in HIF-1α/VEGFA expression. The results showed that, compared with the WT group, the VEGFA and HIF-1α protein levels were signi cantly decreased at 96 hours after ischemia in Gucy1a3-KO mice, suggesting that, mechanistically, the HIF-1α/VEGFA-dependent signaling may mediate the effects of GUCY1A3 on angiogenesis.
Several limitations of this study should be considered. First, in the current study, only silencing experiments were performed. In the next set of experiments, overexpression of the Gucy1a3 gene should be employed to investigate the impact of GUCY1A3 on angiogenesis and cerebral injury after ischemic stroke. Second, a previously published paper suggested that hypertension and the responsiveness to NO in sGCα1 knockout mice are gender-speci c [33] . Therefore, female Gucy1a3-KO mice should be investigated in the future. Third, compensatory mechanisms in the Gucy1a3-KO model, including a compensatory increase in GUCY1A2 expression in Gucy1a3-KO mice, were not evaluated in the present study. Nevertheless, other studies have con rmed that loss of one of the sGC isoforms was not compensated by an upregulation of the remaining one, indicating that deletion of the sGCα1 subunit is not compensated by upregulation of the other form [33,34] .
In conclusion, we demonstrated that GUCY1A3 expression after ischemic stroke may play a signi cant role in neurological function recovery, which is related to angiogenesis in the peri-infarct region. The bene cial effects of GUCY1A3 might be mediated by the HIF-1α/VEGFA signaling pathway. This raises the possibility of targeting Gucy1a3 as a reasonable and novel therapeutic strategy for ischemic stroke.

Declarations
Wu, Xiaoling Li, Jiaoxing Li. The rst draft of the manuscript was written by Man Luo and all authors commented on previous versions of the manuscript. Wenli Sheng contributed to design, analysis and interpretation of data and editing and approving the nal manuscript. All authors read and approved the nal manuscript.

Data Availability
The datasets generated or analysed during the current study are not publicly available due to the co-rst author Dongcan Mo is preparing his master's thesis, this research is a part of the master's thesis but is available from the corresponding author on reasonable request.

Ethics approval
All animal experiments were performed in accordance with approved protocols of the Sun Yat-sen University Animal Care and Use Committee and were in line with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health in China.

Consent for Publication
Not applicable.

Consent to Participate
Not applicable.   Angiogenesis was signi cantly decreased in Gucy1a3-KO mice following ischemic stroke (a) Representative images of immunostaining of CD31 as a vascular density marker. Scale bar: 100 μm.
(b) Quantitative analysis showed that the vascular density was signi cantly decreased in the perifocal striatum of Gucy1a3-KO mice (n=6 per group). Data are expressed as the means ± SD. **P<0.01

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. GUCY1A3KOmiceSupplementary.docx