Balancing vascular regrowth and stabilization after ischemia through CXCR3 signaling

Investigator: Jihui Lee

Supervisor: Amber Stratman

Institution: Washington University in St. Louis

1. Overview

1.1 Scientific Rationale and Importance:

Stroke is the second leading cause of death worldwide and a major contributor to long-term disability and economic burden ^1,2. Ischemic stroke (IS), which accounts for the majority of cases, results from arterial occlusion in the brain and causes tissue injury due to abruptly impaired blood flow. Although acute intervention, such as thrombolysis or mechanical thrombectomy, can restore vessel patency, there are currently no effective therapies that actively promote tissue and vascular regeneration during the post-ischemic recovery phase ^2-5. Consequently, incomplete and dysfunctional vascular repair remains a major barrier to functional tissue recovery after ischemic injury.

Importantly, effective recovery from ischemia requires more than angiogenic vessel growth. The formation of stable, mature, and perfused vascular networks is essential for restoring oxygen and nutrient delivery and for providing instructive cues that support tissue repair ^6-8. Poorly organized or unstable neovessels may fail to reperfuse damaged tissue, exhibit increased permeability, and ultimately impair recovery. Despite the importance of vascular quality, the molecular mechanisms that coordinate endothelial regeneration with vessel stabilization following ischemic injury remain poorly defined.

Blood flow is a fundamental regulator of vascular remodeling, shaping endothelial behavior during injury and recovery. However, how hemodynamic cues integrate vascular growth with subsequent stabilization remains unclear. This proposal will address this critical gap by investigating CXCR3 as a candidate flow-responsive regulatory checkpoint that governs the quality—not merely the extent—of vascular regeneration following ischemic injury.

1.2 Confirmation of funding/facilities/ethics approvals:

Required equipment, facilities, and core services for animal housing and care, RNA sequencing, and confocal imaging are available at Washington University School of Medicine (WUSM) for the proposed project. Ethics approvals for the use of vertebrate animals are already approved.

1.3 Estimated timeline:

Month 1-2: Subaim 1 - Determine the effect of CXCR3 loss on productive or dysfunctional endothelial regeneration following ischemic injury.

Month 2-3: Subaim 2 - Determine the role of CXCR3 in pericyte recruitment and vascular stabilization during post-ischemic recovery.

Month 3-6: Subaim 3: Identify CXCR3-dependent endothelial programs that distinguish productive from non-productive vascular regeneration after ischemic injury.

1.4 Data Sharing Commitment: All analysis data will be shared in a public repository, and the preprint will be uploaded to ResearchHub and bioRxiv.

2. Introduction

2.1 Background and Hypothesis

Restoration of vascular structure and function is a central determinant of tissue repair following ischemic injury. In the brain, ischemia triggers a robust angiogenic response characterized by endothelial sprouting and vascular expansion ^9,10. However, extensive evidence indicates that this response is frequently uncoupled from the formation of stable and functional vascular networks ^6-8. Newly formed vessels may have immature morphology, insufficient mural cell coverage, and increased permeability, limiting effective reperfusion and contributing to persistent tissue dysfunction. Thus, a major unresolved challenge in post-ischemic repair is understanding how endothelial regeneration is coordinated with vessel maturation and stabilization.

Blood flow serves as a key instructive signal that regulates endothelial behavior, including cell polarity, junctional organization, gene expression, and interactions with perivascular cells. During ischemia and subsequent reperfusion, endothelial cells (ECs) experience profound and dynamic changes in shear stress. Although several flow-responsive pathways have been implicated in vascular remodeling, the mechanisms by which hemodynamic cues can integrate vascular regeneration and stabilization remain incompletely understood.

Chemokine signaling pathways are involved in vascular remodeling; however, their roles in integrating endothelial growth with perivascular support during regeneration remain unclear ^11-16. CXCR3, a chemokine receptor best known for its roles in immune cell trafficking, has not been systematically studied in the context of ischemia-induced vascular regeneration ^17-21

My preliminary studies in zebrafish identified CXCR3 as a novel flow-responsive regulator of vascular remodeling ²². Using EC-specific Translating Ribosome Affinity Purification (TRAP) RNA sequencing, I found that cxcr3 expression is induced under no flow conditions, a finding validated by qRT-PCR and HCR RNA-FISH (Pilot Data; Fig. 1) ²³. These results showed that CXCR3 is dynamically regulated by hemodynamic forces. Notably, cxcr3 mutant zebrafish have increased endothelial vascular network formation accompanied by reduced pericyte recruitment, suggesting that CXCR3 signaling is required to constrain endothelial overgrowth while promoting vascular stabilization (Pilot Data; Fig 2-3). These findings highlight a critical knowledge gap: does vascular regeneration in the absence of CXCR3 improve or compromise functional vascular recovery following ischemic injury? Based on these data, my central hypothesis is that low CXCR3 levels are essential for coordinating ischemia-induced vascular expansion, and following reperfusion, CXCR3 levels rise to promote vascular maturation and functional tissue repair. Specifically, I propose that loss of CXCR3 will accelerate vascular regrowth, but if sustained, it will compromise vessel stabilization.

2.2 Specific Hypotheses

H1: Loss of CXCR3 accelerates early endothelial regrowth following ischemic injury.

H2: CXCR3 deficiency impairs pericyte recruitment and vascular stabilization during recovery.

H3: CXCR3 regulates a distinct endothelial translational program that distinguishes productive from non-productive vascular regeneration.

2.3 Impact

Addressing the gap is significant because excessive but poorly stabilized neovascularization can lead to impaired perfusion, sustained permeability, and incomplete tissue repair. Determining how CXCR3 regulates post-ischemic vascular regeneration will clarify how flow- and injury-responsive signaling pathways govern the quality of neovascular growth. Importantly, this work will establish CXCR3 signaling as a regulatory checkpoint that coordinates endothelial expansion with pericyte recruitment, a process essential for generating stable and functional blood vessels. Overall, the proposed research will advance fundamental understanding of vascular regeneration mechanisms and identify CXCR3 as a potential regulator of functional neovascularization following ischemic injury. These insights may inform future therapeutic strategies aimed at enhancing vascular repair and regeneration in response to disease.

3. Methods

This project will test whether CXCR3 functions as a regulatory checkpoint that balances vascular expansion with stabilization after ischemic injury. Through genetically mutated zebrafish models combined with focal photothrombotic injury, high-resolution live imaging, and EC-specific translational profiling, I will assess how CXCR3 activity shapes the structural and functional quality of regenerating vessels within a 6-month funding period ^23-25.

Aim: Determine how CXCR3 regulates vascular maturation and functional recovery following ischemic injury.

Working hypothesis: CXCR3 deficiency leads to accelerated but poorly stabilized vascular regeneration following ischemic injury, resulting in impaired functional tissue recovery.

Subaim 1: Determine the effect of CXCR3 loss on productive or dysfunctional endothelial regeneration following ischemic injury.

Sample: Focal cerebrovascular injury will be induced inEC–labled transgenic zebrafish (Tg(fli1a:eGFP)). Wild-type and cxcr3 mutant zebrafish embryos maintained on Tg(fli1a:eGFP) background will be compared (n=20 embryos per genotype, pooled from ≥3 independent clutches).

Procedures:

Focal ischemic induction: Localized cerebrovascular damage will be generated to defined vessels (e.g., dorsal longitudinal vein or central arteries) using Rose Bengal-mediated photothrombosis ^24,25. Following systemic Rose Bengal injection, spatially restricted light exposure will induce targeted vessel occlusion and cessation of local blood flow. Cerebral blood flow and vascular recovery will be monitored using intravascular fluorescent tracers at defined time points (6, 24, 48, 72 hours post-injury), capturing both acute and recovery phases through time-lapse confocal imaging.

Planned Analyses:

Structural regeneration

Endothelial regeneration in wild-type and cxcr3 mutant animals will be quantified using:

Regrowth kinetics (time to revascularization of injured region)
Vessel density (total EC-positive area per defined ROI)
Branch point number (network complexity)

Functional recovery

Productive regeneration will be defined as restoration of perfused, organized vascular networks, whereas dysfunctional regeneration will be characterized by excessive angiogenesis without effective reperfusion. Functional recovery will be assessed by:

Perfusion restoration (fluorescent tracer circulation within regenerated vessels)
Blood flow velocity (particle tracking analysis)
Vessel integrity (quantification of tracer leakage area)

Statistical Analysis: Data will be tested for normality. For comparisons between genotypes across time points, a two-way ANOVA (genotype × time) will be performed, followed by Tukey’s post hoc test for multiple comparisons. For single time-point comparisons, unpaired two-tailed Student’s t-test (or Mann–Whitney U test for non-parametric data) will be used.

Subaim 2: Determine the role of CXCR3 in pericyte recruitment and vascular stabilization during post-ischemic recovery.

Sample: Focal ischemia will be induced in wild-type and cxcr3 mutant zebrafish embryos crossed onto double transgenic endothelial and pericyte reporter backgrounds ([Tg(fli1a:eGFP)]; [Tg(pdgfrb:gal4/UAS:RFP)]), enabling simultaneous in vivo visualization of ECs and pericytes. A total of n=20 embryos per genotype (pooled from ≥3 independent clutches) will be analyzed per experimental condition.

Procedures:

Focal ischemic induction: Focal cerebrovascular injury will be induced using the Rose-Bengal-mediated photothrombotic model as described in Subaim 1.

Live imaging and recovery monitoring: Cerebral blood flow and vascular recovery will be monitored at 6, 24, 48, and 72 hours post-injury (hpi). Intravascular fluorescent tracers will be injected to assess blood flow restoration and vascular permeability. Time-lapse confocal imaging will capture both acute and recovery phases.

Planned Analyses:

Pericyte recruitment

Pericyte coverage and recruitment dynamics will be quantified using:

Pericyte coverage index (pericyte-covered vessel length / total regenerated vessel length)
Pericyte number per defined ROI
Temporal dynamics of pericyte recruitment (6-72 hpi)

Vascular stabilization and integrity

Vessel diameter normalization over time
Barrier integrity (quantified as tracer leakage area)

Statistical Analysis: Data distribution will be tested for normality. For comparisons between genotypes across time points, a Two-way ANOVA (genotype × time) followed by Tukey’s post hoc test will be used to compare pericyte coverage and stabilization metrics across time points. For single time-point comparisons, unpaired two-tailed Student’s t-test (or Mann–Whitney U test for non-parametric data) will be applied. Analyses will be performed blinded to genotype, and data will be presented as mean ± SD (p < 0.05 considered significant).

Subaim 3: Identify CXCR3-dependent endothelial programs that distinguish productive from non-productive vascular regeneration after ischemic injury.

Working hypothesis: CXCR3 regulates a distinct endothelial gene program that coordinates functional vascular regeneration.

Sample: EC-specific translational profiling will be performed using transgenic zebrafish (Tg(flk1:AngioTag)) crossed into wild-type and cxcr3 mutant backgrounds. This system enables immunoprecipitation of ribosome-bound mRNA specifically from ECs in vivo ²³.

For each genotype and condition (injury vs. uninjured control), n=200 embryos per group will be collected at defined post-injury time points (e.g., 24 and 48 hpi) corresponding to active regeneration and stabilization phases.

Procedures:

EC-specific RNA isolation: Following focal ischemic injury (as described in Subaim 1-2), embryos will be harvested and endothelial ribosome-bound transcripts isolated using TRAP method. RNA will be extracted and subjected to RNA sequencing.

Differential gene expression analysis: Translational profiles from wild-type and cxcr3 mutant ECs will be compared under ischemic conditions to identify CXCR3-dependent pathways involved in:

Endothelial sprouting and migration
Vessel maturation and junctional stabilization
Pericyte recruitment
Flow-responsive signaling programs

Functional pathway enrichment analyses (Gene ontology and KEGG) will be performed to define functional gene networks associated with productive versus non-productive vascular regeneration.

Planned Analyses:

Integration with vascular phenotypes

Identified molecular signatures will be integrated with structural and functional vascular phenotypes defined in Subaim 1 and 2. Correlation analysis will determine whether specific gene modules are associated with:

Stable, perfused vascular recovery (productive regeneration)
Excessive but poorly stabilized network expansion (non-productive regeneration)

Functional validation

Top candidate CXCR3-dependent regulators will be prioritized based on fold change, pathway relevance, and association with functional phenotypes. Functional relevance will be tested using:

CRISPR/Cas12-mediated gene disruption
Pharmacologic pathway modulation

Following ischemic injury, vascular stabilization, pericyte recruitment, permeability, and blood flow recovery will be quantified as described in Subaim 1–2.

Ethics and Data Management

Ethics approvals for the use of vertebrate animals are already approved.

All analysis data will be shared in a public repository, and the preprint will be posted on ResearchHub and bioRxiv.

4. Pilot Data

These pilot data provide preliminary evidence that CXCR3 is dynamically regulated by hemodynamic forces and functionally influences the vascular network and pericyte recruitment. These findings directly support the central hypothesis that CXCR3 acts as a regulatory checkpoint coordinating endothelial expansion with vascular stabilization during post-ischemic regeneration.

Figure 1. The chemokine ligand, cxcl11, and its receptor cxcr3, are regulated by blood flow in ECs during development. (A) Protocol schematic. (B) Data from TRAP mRNA isolation and sequencing. (C) qRT-PCR validation of sequencing datasets. (D) HCR-FISH of the zebrafish cranial vasculature. kdrl (endothelial cell marker, gray), cxcr3.1 (magenta), and cxcr3.3 (green)

Figure 2. CXCR3 inhibits cranial angiogenesis in the developing zebrafish. (A) Representative confocal images of cranial vascular networks at 72 hpf in WT or homozygous cxcr3.1^Δ7/Δ7 cxcr3.2^Δ2Δ2 cxcr3.3^Δ8Δ8triple mutant animals. Scale bar=250 µm. (B) Quantification of the vascular area normalized to the WT control average.

Figure 3. CXCR3 promotes pericyte recruitment to blood vessels in vivo. (A) Representative images from the Tg(fli1a:GFP); Tg(pdgfrb:Gal4FF; UAS:RFP) double transgenic zebrafish line, marking endothelial cells in magenta and pericytes in green. Arrows point to pericytes that are associated with the cranial vasculature in 72 hpf zebrafish. (B) Representative images and quantification of the cxcr3.1^Δ7/Δ7 cxcr3.2^Δ2/Δ2 cxcr3.3^Δ8/Δ8 triple mutants vs triple heterozygous controls on the Tg(pdgfrb:gal4ff); (UAS:GFP) transgenic line, marking pericytes in green. Vascular networks were labeled by injection of Evans blue into the sinus venosus at 72 hpf. Quantification of the number of pericytes associated per 100 µm of the vessels in zebrafish forebrain.

5. Budget

6. References

1. Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments. Neuron. 2010;67:181-198. doi: 10.1016/j.neuron.2010.07.002

2. Feigin VL, Brainin M, Norrving B, Martins SO, Pandian J, Lindsay P, M FG, Rautalin I. World Stroke Organization: Global Stroke Fact Sheet 2025. Int J Stroke. 2025;20:132-144. doi: 10.1177/17474930241308142

3. Hankey GJ, Blacker DJ. Is it a stroke? BMJ. 2015;350:h56. doi: 10.1136/bmj.h56

4. Wang L, Xiong X, Zhang L, Shen J. Neurovascular Unit: A critical role in ischemic stroke. CNS Neurosci Ther. 2021;27:7-16. doi: 10.1111/cns.13561

5. Hankey GJ. Stroke. Lancet. 2017;389:641-654. doi: 10.1016/S0140-6736(16)30962-X

6. Hatakeyama M, Ninomiya I, Kanazawa M. Angiogenesis and neuronal remodeling after ischemic stroke. Neural Regen Res. 2020;15:16-19. doi: 10.4103/1673-5374.264442

7. Ergul A, Alhusban A, Fagan SC. Angiogenesis: a harmonized target for recovery after stroke. Stroke. 2012;43:2270-2274. doi: 10.1161/STROKEAHA.111.642710

8. Liu J, Wang Y, Akamatsu Y, Lee CC, Stetler RA, Lawton MT, Yang GY. Vascular remodeling after ischemic stroke: mechanisms and therapeutic potentials. Prog Neurobiol. 2014;115:138-156. doi: 10.1016/j.pneurobio.2013.11.004

9. Huang H, Huang Q, Wang F, Milner R, Li L. Cerebral ischemia-induced angiogenesis is dependent on tumor necrosis factor receptor 1-mediated upregulation of alpha5beta1 and alphaVbeta3 integrins. J Neuroinflammation. 2016;13:227. doi: 10.1186/s12974-016-0697-1

10. Zhang ZG, Zhang L, Jiang Q, Zhang R, Davies K, Powers C, Bruggen N, Chopp M. VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest. 2000;106:829-838. doi: 10.1172/JCI9369

11. Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol. 2001;2:108-115. doi: 10.1038/84209

12. Stratman AN, Burns MC, Farrelly OM, Davis AE, Li W, Pham VN, Castranova D, Yano JJ, Goddard LM, Nguyen O, et al. Chemokine mediated signalling within arteries promotes vascular smooth muscle cell recruitment. Commun Biol. 2020;3:734. doi: 10.1038/s42003-020-01462-7

13. Weber C, Schober A, Zernecke A. Chemokines: key regulators of mononuclear cell recruitment in atherosclerotic vascular disease. Arterioscler Thromb Vasc Biol. 2004;24:1997-2008. doi: 10.1161/01.ATV.0000142812.03840.6f

14. Fujita M, Cha YR, Pham VN, Sakurai A, Roman BL, Gutkind JS, Weinstein BM. Assembly and patterning of the vascular network of the vertebrate hindbrain. Development. 2011;138:1705-1715. doi: 10.1242/dev.058776

15. Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawabata K, Kataoka Y, Kitamura Y, Matsushima K, Yoshida N, Nishikawa S, et al. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature. 1998;393:591-594. doi: 10.1038/31261

16. Takabatake Y, Sugiyama T, Kohara H, Matsusaka T, Kurihara H, Koni PA, Nagasawa Y, Hamano T, Matsui I, Kawada N, et al. The CXCL12 (SDF-1)/CXCR4 axis is essential for the development of renal vasculature. J Am Soc Nephrol. 2009;20:1714-1723. doi: 10.1681/ASN.2008060640

17. Groom JR, Luster AD. CXCR3 in T cell function. Exp Cell Res. 2011;317:620-631. doi: 10.1016/j.yexcr.2010.12.017

18. Campanella GS, Tager AM, El Khoury JK, Thomas SY, Abrazinski TA, Manice LA, Colvin RA, Luster AD. Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria. Proc Natl Acad Sci U S A. 2008;105:4814-4819. doi: 10.1073/pnas.0801544105

19. Hancock WW, Lu B, Gao W, Csizmadia V, Faia K, King JA, Smiley ST, Ling M, Gerard NP, Gerard C. Requirement of the chemokine receptor CXCR3 for acute allograft rejection. J Exp Med. 2000;192:1515-1520. doi: 10.1084/jem.192.10.1515

20. Mikucki ME, Fisher DT, Matsuzaki J, Skitzki JJ, Gaulin NB, Muhitch JB, Ku AW, Frelinger JG, Odunsi K, Gajewski TF, et al. Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints. Nat Commun. 2015;6:7458. doi: 10.1038/ncomms8458

21. Panzer U, Steinmetz OM, Paust HJ, Meyer-Schwesinger C, Peters A, Turner JE, Zahner G, Heymann F, Kurts C, Hopfer H, et al. Chemokine receptor CXCR3 mediates T cell recruitment and tissue injury in nephrotoxic nephritis in mice. J Am Soc Nephrol. 2007;18:2071-2084. doi: 10.1681/ASN.2006111237

22. Lee J, Goeckel ME, Levitas A, Colijn S, Shin J, Hindes A, Mun G, Burton Z, Chintalapati B, Yin Y, et al. CXCR3-CXCL11 Signaling Restricts Angiogenesis and Promotes Pericyte Recruitment. Arterioscler Thromb Vasc Biol. 2024;44:2577-2595. doi: 10.1161/ATVBAHA.124.321434

23. Miller MF, Greenspan LJ, Gildea DE, Monzo K, Margolin G, Pham VN, Ameyaw KK, Price L, Aloi N, Stratman AN, et al. In vivo profiling of the endothelium using 'AngioTag' zebrafish. Angiogenesis. 2025;28:40. doi: 10.1007/s10456-025-09990-8

24. Chen J, He J, Ni R, Yang Q, Zhang Y, Luo L. Cerebrovascular Injuries Induce Lymphatic Invasion into Brain Parenchyma to Guide Vascular Regeneration in Zebrafish. Dev Cell. 2019;49:697-710 e695. doi: 10.1016/j.devcel.2019.03.022

25. Nayaka N, Adnyana IK, Anggadiredja K, Wibowo I. Drug screening for ischemic stroke using larvae and adult zebrafish model: a review. Lab Anim Res. 2025;41:1. doi: 10.1186/s42826-024-00232-4

Balancing vascular regrowth and stabilization after ischemia through CXCR3 signaling

Balancing vascular regrowth and stabilization after ischemia through CXCR3 signaling

Funding Progress

Funders

Peer Reviews

Related Funding

Request for Proposals - Vascular Biology

Nonprofit Recipient

Topics

DOI