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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Curr Opin Hematol. 2021 May 1;28(3):179–188. doi: 10.1097/MOH.0000000000000649

Mouse models of vascular development and disease

Ondine Cleaver 1
PMCID: PMC8281369  NIHMSID: NIHMS1713295  PMID: 33769415

Abstract

Purpose of review

The use of genetic models has facilitated the study of the origins and mechanisms of vascular disease. Mouse models have been developed to specifically target endothelial cell (EC) populations, with the goal of pinpointing when and where causative mutations wreck their devastating effects. Together, these approaches have propelled the development of therapies by providing an in vivo platform to evaluate diagnoses and treatment options. This review summarizes the most widely used mouse models that have facilitated the study of vascular disease, with a focus on mouse models of vascular malformations (VMs) and the road ahead.

Recent findings

Over the past three decades, the vascular biology scientific community has been steadily generating a powerful toolkit of useful mouse lines that can be used to tightly regulate gene ablation, or to express transgenic genes, in the murine endothelium. Some of these models inducibly (constitutively) alter gene expression across all ECs, or within distinct subsets, by expressing either Cre recombinase (or inducible versions such as CreERT), or the tetracycline controlled transactivator protein tTA (or rtTA). This now relatively standard technology has been used to gain cutting edge insights into vascular pathologies, by allowing in vivo modeling of key molecular pathways identified as dysregulated across the vast spectrum of vascular anomalies, malformations and dysplasias. However, as sequencing of human patient samples expands, the number of interesting candidate molecular culprits keeps increasing. Consequently, there is now a pressing need to create new genetic mouse models to test hypotheses and to query mechanisms underlying vascular disease.

Summary

This review assesses the collection of mouse driver lines that have been instrumental is identifying genes required for blood vessel formation, remodeling, maintenance/quiescence and disease. In addition, the usefulness of these driver lines is underscored here by cataloguing mouse lines developed to experimentally assess the role of key candidate genes in vascular malformations (VMs). Despite this solid and steady progress, numerous new candidate VM genes have recently been identified for which no mouse model yet exists.

Keywords: Mus musculus (mouse), vasculature, vascular disease, endothelial cell (EC), transgenic mice, Cre recombinase, vascular malformations (VMs), cerebral cavernous malformations (CCMs)

INTRODUCTION

In the field of vascular biology, animal models have played a vital role in the discovery of fundamental mechanisms underlying blood vessel health, and in developing therapeutics for a wide range of diseases. Many models of cardiovascular diseases have been developed over the decades in larger animal models, such as rats and swine, using surgical or pharmacological interventions. However, mouse as a model system offers many clear advantages, including the power of genetics. Additional advantages include ease of breeding, large size of litters, availability of multiple inbred strains, ease of genetic manipulation, low cost of maintenance, smaller amounts of pharmacological drugs needed for studies, and shorter timeframe as some diseases develop rapidly in mice. As a consequence, genetically modified mice have been widely used, powerful tools to help elucidate the roles of specific genes and signaling pathways in a wide variety of biological vascular processes, including blood vessel formation, remodeling, quiescence and integrity. Murine models have also been cleverly “customized” and engineered to mimic specific genetic mutations first identified in human patients. The mouse versions provide efficient and tractable platforms for testing therapeutic interventions, in the effort to target aberrant signal transduction pathways.

Here, we review key mouse model tools in the field and their impact on the study of one group of vascular diseases called vascular malformations (VMs).

MOUSE MODELS THAT GENETICALLY TARGET THE VASCULATURE

Our knowledge of vascular biology has been immensely influenced by our ability to carry out both loss-of-function (LOF) and gain-of-function (GOF) genetic experiments in mouse models. These tools, created over the last few decades by individual investigators, have allowed dissection of genetic pathways that underlie both vascular development and disease. Genetic mouse models allow ablation of specific genes in endothelial or smooth muscle cells of the vasculature, as well as in specific subsets thereof. In addition, when inducible, these models allow temporal fine tuning of gene ablation, thereby revealing spatiotemporal requirements for various signaling pathways in distinct blood vessel cell types.

Many such conditional knockout (cKO) mice, also called inducible knockout (iKO) mice, are based on Cre-mediated excision (Table 1). Others employ doxycycline (dox) regulated Tet-On and Tet-Off expression systems to drive either silencing or overexpression of selected genes. The specificity of each driver line depends entirely on the promoters selected to drive these regulatory cassettes (Cre/CreERT/tRA/rtTA). For instance, the endogenous locus promoter of an endothelial gene, or large fragments containing both promoters and enhancers, often contain all the regulatory elements to drive faithful cell specific expression. These regulatory elements dictate how, when and where a transgene will be expressed. These transgenic tools, once cutting edge, are now a mainstay of biomedical science. However, interpretation of resulting phenotypes acquired using mouse models requires consideration of timing, penetrance and context. In this issue, Garcia-Gonzales and Benedito discuss in detail the caveats, both positive and negative, regarding the efficacy of conditional genetic approaches.

TABLE 1. Cre and Tet lines useful to manipulate gene expression in mouse ECs.

Non-exhaustive list of available transgenic lines that drive expression of Cre or tTA/rtTA in the mouse endothelium, either pan-endothelial or in different EC subpopulations. (Table adapted from Payne et al., 2018 [1] and Assmann et al., 2016 [2]). (Endothelial cell, EC; Expressed in all ECs, Pan EC).

Mouse Driver Line Source of available line Expression in ECs Activity initiation EC restricted Reference
Acvrl1-Cre
(Alk1-Cre, L1cre)		 Tg(Acvrl1-cre)B1Spo
MGI:4438606		 Brain ECs, lung ECs, Widespread peripheral ECs E10.5 yes [3]
Apln-CreERT2 Aplntm1.1(cre/ERT2)Bzsh
MGI:5637737		 Sprouting ECs E10.5 yes [4]
Aplnr-CreERT2
(Apj)		 B6.Cg-Tg(Tek-cre)1Ywa/J
JAX stock #008863		 Sinus venosus, capillaries, vein ECs E10.5 yes [5]
Bmx-CreERT2 Tg(Bmx-cre/ERT2)1Rha
MGI:5513853		 Subset of arterial ECs E10.5 yes [6]
Cdh5-Cre
(VE-cad)		 Tg(Cdh5-cre)7Mlia
JAX stock #006137		 Pan EC, some HSCs E7.5 no [7]
Cdh5-CreERT2
(VE-cad)		 Tg(Cdh5-cre/ERT2)1Rha
Taconic # 13073-M		 Pan EC E9.5 yes [8]
Cdh5-tTA
(VE-cad)		 Tg(Cdh5-tTA)D5Lbjn
JAX stock #013585		 Pan EC, retina - no [9]
Esm1-CreERT2 Tg(Esm1-cre/ERT2)1Rha
MGI:5513853		 Tip ECs P4 yes [10]
Fabp4-Cre
(Ap2)		 Tg(Fabp4-cre)1Rev
JAX stock #018965		 Coronary ECs, adipose tissue, macrophages E16.5 no [11]
Fabp4-CreERT2
(Ap2)		 Tg(Fabp4-cre/ERT2)1Ipc
MGI:2387425		 Coronary ECs, adipose tissue, macrophages E16.5 no [12]
Flk-1
(VEGFR2, Kdr)		 Kdrtm1(cre)Sato
JAX stock #018977		 Pan EC, HSCs, skeletal muscle E8.5 no [13]
Mfsd2a-CreERT2 Mfsd2aem1(cre/ERT2)Bzsh
MGI:5909986		 Brain blood vessels, hepatocytes, placenta, colon - no [14]
Mx1-Cre Tg(Mx1-cre)1CgnJ
JAX stock #003556 		Brain ECs, liver, lymphocytes, interferon induced - no [15]
Nfatc1-Cre Nfatc1tm1.1(cre)Bz
MGI:5471107 		Endocardial ECs, hair follicle, osteoblasts, macrophages E9.0 no [16]
Nfatc1-CreERT2 Nfatc1tm1.1(cre/ERT2)Bzsh
MGI:5637438 		Endocardial ECs, sinus venosus ECs E8.5 yes [17]
Npr3-CreERT2 Npr3tm1.1(cre/ERT2)Bzsh
MGI:5804184		 Endocardial ECs, subset of epicardial cells E8.5 no [18]
Pdgfb-CreERT2 Tg(Pdgfb-icre/ERT2)1Frut
MGI:3793852 		Capillary ECs, arterial ECs, keratinocytes, megakaryocytes E9.5 no [19]
SCL-CreERT2
(Tal1-CreERT2)		 Tg(Tal1-cre/ERT)42-056Jrg
MGI:3579158		 Embryonic ECs, Tumor ECs - no [20]
Sftpa1 Tg(Sftpa1-cre)1Xya
MGI:3696659		 Brain ECs, lung, stomach - no [21]
Slco1c1(BAC)-CreERT2
(OATP14)		 Tg(Slco1c1-icre/ERT2)1Mrks
MGI:5301361		 Brain ECs, some lung - no [22]
Sox17-Cre Sox17tm1(iCre)Heli
MGI:3852645		 Subset of arterial ECs, Thymus, pancreas, other organs E8.5 no [23]
Tie1-Cre Tg(Tie1-cre)9Ref
MGI:2385916		 Pan EC, yolk sac, placenta E8,5 no [24]
Tie2-Cre
(Tek)		 Tg(Tek-cre)1Ywa
JAX stock #008863		 Pan EC, Heart valves, HSCs E7.5 no [25]
Tie2-CreERT2
(Tek)		 Tg(Tek-cre/ERT2)1Soff
MGI:3837451		 Pan EC - yes [26]
Tie2-rtTA;TRE-lacZ
(Tek)		 Tg(Tek-rtTA,TRE-lacZ)1425Tpr/J
JAX stock #005493		 Pan EC - no [27]
Tie2-tTA
(Tek)		 Tg(Tek-tTA)1Rwng
MGI:4431194		 Pan EC, liver, uterus E10.5 no [28]
vWF-Cre Tg(VWF-Cre)1304Roho
MGI:5319174		 Brain EC - [29]
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Here, we list some commonly used mouse driver lines that have been instrumental is identifying genes required for blood vessel development, remodeling, maintenance/quiescence and disease.

MOUSE MODELS OF VASCULAR MALFORMATIONS:

Vascular malformations (VMs) comprise a highly diverse group of vascular anomalies (VAs) that are classified by the type of vessel affected, which can be capillaries, veins, arteries, lymphatic vessels, or any combination thereof. Arteriovenous malformations (AVMs) are a subset of VMs that often cause abnormal “high-flow” (high blood flow) connections between arteries and veins, and can displace capillaries with enlarged, tortuous vessels. When they occur in the brain, these brain AVMS (BAVMs) can rupture, sometimes causing life threatening cranial hemorrhages. Outside the brain, AVMs can cause hemorrhages in skin or other organs, resulting in deformities or chronic pain. Other VMs are “low-flow”, involving aberrant venous (venous malformations), capillary (capillary malformations, CMs) or lymphatic vessels (lymphatic malformations (LMs). These types of VMs are the most common subcutaneous lesions and are often located in the head and neck. However, lesions can grow, become fluid-filled and painful, and even hinder movement. A subset of brain VMs are the Cerebral Cavernous Malformations (CCMs). These represent a rare but debilitating neurovascular disease that displays enlarged and tortuous cranial blood vessels that can rupture in a life threatening manner, leading to seizures, neurological deficits or death.

Unfortunately, at this point in time, therapeutic options for these conditions remain very limited. Current treatment for AVMs are limited to surgery, radiosurgery and embolization, and are often mostly effective in smaller lesions. Clinical approaches for low-flow VMs include percutaneous drainage, sclerotherapy (injecting solutions into a vessel), laser therapy and surgery. The primary treatment option for CCMs is MRI monitoring and surgical removal, as radiation has not been shown to be effective. Given the limited therapeutic options, it is clear that a deeper molecular and pathophysiological understanding is urgently needed. New treatment possibilities will depend on the discovery of inherited and somatic genetic mutations, for which pathways can be explored and tested for useful interventions.

The field of VM research has indeed grown rapidly in recent years, spurred by the identification of numerous molecular pathways and genetic mutations found to be associated with the formation and progression of patient lesions.[30] Single cell sequencing analyses of both endothelial and vascular mural cells have revealed an unexpected heterogeneity in VMs lesions, painting an complex portrait of their etiology.[31, 32] It is believed that many VMs form due to aberrant vascular development, which likely occurs early during embryogenesis. However, sporadic VMs may arise from a combination of underlying congenital mutations (found in all cells), which are later compounded by a local “second hit” spontaneous mutation specially in ECs. These spatiotemporal complexities of VM lesion formation are an area of active current research for which new genetic mouse models are needed.

Here, we briefly review several types of VMs found in human patients, focusing on the approaches used to study them, the therapeutic options, and useful murine models to study them, when available.

ARTERIOVENOUS MALFORMATIONS (AVMs)

Arteriovenous malformations (AVMs) are aberrant vessels that lose their normal hierarchical organization of artery-capillary-vein. Blood flow in AVMs stream arterial blood directly into veins via arteriovenous (AV) shunts, bypassing entirely any normal capillary connections. Vessels in AVMs are usually dilated, tortuous and may connect via a tangle of abnormal vessels, or a nidus. AVMs can be found anywhere in the body, but when in the brain or spinal cord, they present substantial risks upon rupture. One third of AVMs have no safe current treatments.

Animal models have been developed to better elucidate AVM pathophysiology and develop therapeutic approaches. Efforts at replicating AVM hemodynamics to better study them has led to a number of surgical approaches in large animal models, such as swine, sheep and rats, where the goal was to create AVMs via anastomosis of the jugular and carotid arteries.[33] Identification of underlying genetic mutations associated with AVMs in patient samples, however, has opened the door to the development of genetic mouse models of AVMs. The possibility of going beyond AVM surgery or embolization, and possibly use approaches such as gene therapy or targeted pharmacological interventions to treat large AVMs, has accelerated the desire to understand these lesions at the molecular level.

To date, no single animal model, either surgical or genetic, has fully mimicked human AVMs, as each model has displayed distinct limitations. However, mouse models modulating key signaling pathways, such as the Notch and TGFβ pathways, have contributed important insights into AVM formation (Table 2). The hope is that genetic mouse models will together, in time, further dissect the pathways dysregulated in AVMs and expand our understanding of their etiology. Below, Table 2 lists mouse models developed to investigate key genetic candidates associated with AVMs.

TABLE 2. Genetic mouse models useful to study vascular malformations.

Non-exhaustive list of mouse models of Arteriovenous Malformations (AVMs), Brain AVMs (BAVMs), Lymphatic malformations (LMs), Hereditary Hemorrhagic Telangiectasias (HHTs) and Cerebral Cavernous Malformations (CCMs). Table adapted from Nielsen et al., 2016 [49], Tual-Chalot et al, 2015 [34], Zeng et al., 2019[50] and McDonald et al., 2011[39]. (Endothelial cell, EC; lymphatic endothelial cell, LEC; overexpression, OE; vascular endothelial growth factor, VEGF; vascular smooth muscle cell, vSMC).

Mouse Driver Line Genetic manipulation Site of vascular defect Vascular anomaly Reference
Acvlr1 +/−
(Alk1)		 Embryonic germline global heterozygous deletion Skin, oral, lung, liver, gut AVMs in adults AVM, HHT [51]
Acvlr1flox/flox
globalKO
R26R-CreER		 Conditional KO (cKO) in all cell types Gut and lung hemorrhage, skin AVMs upon wounding. AVM, HHT [52]
Acvlr1 flox/flox
ECKO
L1-Cre		 Conditional KO in ECs of brain, lung and gut AVMs, hemorrhage in brain, lung and gut AVM, HHT [52, 53]
Acvlr1 flox/flox
ECKO Cdh5-Cre-ERT2
(Activin receptor like kinase 1)		 Conditional KO in ECs postnatally Retinal AVMs, lung hemorrhage AVM, HHT [34]
Acvlr1 flox/flox
ECKO
Pdgfb-CreER		 Conditional KO in ECs in adult Brain AVMs upon VEGF stimulation AVM, HHT [54]
Acvlr1 flox/flox
SMCKO
SM22a-CreER		 Conditional KO in ECs and vSMCs Brain and spinal AVMs AVM, HHT [55]
Acvlr1 flox/flox
ECKO
Scl-CreER		 Conditional KO in adults Skin AVMs following wounding, visceral AVMs AVM, HHT [56]
Acvlr1 flox/flox
Ad-Cre +
AAV-VEGF		 Local deletion in adults, with VEGF stimulation BAVM-like in basal ganglia upon Cre and VEGF stimulation AVM, HHT [57]
Cdc42 flox/flox;
Cdh5-CreERT2 		Postnatal deletion of Cdc42 in ECs
Cdc42iΔEC 		BAVMs in venous and perivenous capillaries CCM [58]
Ccm1+/−
(KRIT)		 Global heterozygous deletion of Ccm1 Vascular leakage in brain and lungs - [59]
Ccm1+/−;
Cdh5-CreERT2
(KRIT)		 Postnatal deletion of Ccm1 Brain lesions, EndMT-like traits CCM-like lesions [60]
Ccm1+/−;Trp53−/−
(KRIT)		 Global deletion of both Ccm1 and tumor suppressor mutant Trp53(p53) Vascular malformations, ectatic dilated capillaries, thrombosis CCM [38]
Ccm1+/−;Msh2−/−
(KRIT) 		Global deletion of both Ccm1 and mismatch repair mutant Msh Vascular malformations, dilated capillaries, thrombosis, early and late stage large CCMs CCM [39]
Ccm2+/−
(MGC4607)		 Global heterozygous deletion of Ccm2 Low rate of vascular lesions CCM [61]
Ccm2 flox/flox;
Slc1o1c1-CreERT2
(MGC4607)		 Postnatal deletion of Ccm2 in ECs
BECiCCM2		 Brain lesions, hemorrhages CCM [62]
Ccm3 flox/flox;
Pdgfb-CreERT2
(Pdcd10 ECKO)		 Postnatal deletion of Ccm3 in ECs Brain lesions, hemorrhages CCM [41]
Ccm3 flox/flox;
Slc1o1c1-CreERT2
(Pdcd10 ECKO)		 Postnatal deletion of Ccm3 in brain ECs Brain lesions, hemorrhages CCM [41, 63]
Endoglin +/− Embryonic global heterozygous deletion Eng+/−: Brain AVMs
(Nulls have embryonic yolk sac vascular defects, hemorrhage, heart defects).		 BAVM, HHT [64]
Endoglin flox/flox
ECKO
Cdh5-Cre-ERT2 		Conditional knockout in ECs Neonatoal retinal AVMs, enlarged subdermal veins near VEGF stimulation. AVM, HHT [65]
Endoglin flox/flox
globalKO
R26R-CreER		 Conditional KO in all cell types upon induction AVMs dependent on VEGF stimulation or wounding. AVM, HHT [56, 66]
Endoglin flox/flox
SMCKO
SM22a-CreER T2 		Conditional KO in vSMCs and ECs Scattered AVMs, brain and spinal cord microhemorrhages. AVM, HHT [66]
Endoglin flox/flox
ECKO
Scl-CreER		 Conditional KO in brain ECs Skin AVMs upon wounding. AVM, HHT [56]
Foxn1nu
(athymic nude mice)
JAX stock #002019		 Xenograft of patient VMs with PIK3CA or TIE2 mutations into homozygous nude spontaneous mutation, lack of Tcell immunity Anastomosis of human VMs with host mouse vasculature. - [67]
KrasG12D driven by Slc1o1c1-CreER
(ibEC-KrasG12D)		 Expression of GOF allele of KRAS in postnatal ECs Brain AVMs AVM [68]
Mgp +/− Global deletion of Mgp Postnatal brain AVMs AVM [69]
PIK3CAOE;
T-Cre-ERT2
(MosMes) or
Vegfr3-c		 Knock-in of PIK3CAH1047R in Rosa26 locus expressed in mesoderm or LECs Sporadic VMs when expressed throughout mesoderm

Lymphatic (LMs) when expressed in LECs		 VMs, LMs [70, 71]
PIK3CAOE ;
Prox1-CreERT2 		Knock-in of PIK3CAH1047R in Rosa26 locus expressed in LECs Lymphatic hyperplasia, dysfunction, lymphatic formation in bone LMs [48]
RASA1 +/−
(p120RasGap, RasGap, CM-AVM)		 Global KO of RASA1

Rasa1R780Q mutation		 Vascular developmental defects CM-AVM-like [45, 72]
Rbpj flox/flox ;
Cdh5-Cre-ER T2 		Postnatal deletion of Rbpj Abnormal AV shunts, tortuous vessels AVM like [73]
Smad4 flox/flox ;
Cdh5-Cre-ER T2 		Postnatal deletion of Smad4 Retinal AVMs, likely PAVMs HHT [74]
Tie2-tTA;TRE-Notch4*
(Tek)		 Postnatal expression of constitutively active Notch4 in ECs Liver, uterus and skin AVMs AVM [28]
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HEMORRHAGIC TELANGIECTASIA (HHT)

Hereditary hemorrhagic telangiectasia (HHT) is a genetic autosomal dominant disorder characterized by widespread blood vessel weakness resulting in dilatation of vessels and development of vascular shunts. HHTs are generally associated with small AVMs (telangiectases) of the skin, mucosa or gut, and larger brain, lung or liver AVMs. HHT is also known as Olser-Weber-Rendu disease in humans, and is the most prevalent of AVM disorders. Small mucosal AVMs can result in frequent bleeding, including chronic nosebleeds (epistaxis). Individuals with HHT can develop pulmonary AVMs (PAVMs) leading to low levels of circulating oxygen (cyanosis). Over 600 mutations have been discovered in four genes in human lesions, including endoglin (ENG), activin-like kinase 1 (ACVRL1), the signal transducing factor SMAD4 and bone morphogenetic protein receptor 9 (BMPR9). There is currently no established effective treatment, although management of the disease has evolved towards antiangiogenic approaches to attempt to reduce bleeding, however this has had marginal success.

An interesting theme that has emerged from the growing body of work on HHTs is that the key cell type likely to drive AVM formation is the endothelial cell. Loss of Acvrl1 or endoglin proteins from vascular ECs is required for lesion formation, whereas ablation in other cell types including vascular smooth muscle cells (vSMCs), pericytes, or macrophages does not catalyze significant AVM formation.[34] Below, Table 2 lists mouse models developed to investigate key genetic candidates associated with HHT AVMs.

CEREBRAL CAVERNOUS MALFORMATIONS (CCMS)

Cerebral cavernous malformations (CCMs), or cavernous angiomas, are primarily sporadic vascular malformations that develop in the brain and can lead to debilitating cerebral hemorrhages. CCMs account for 5-15% of all central nervous system VMs, and 1 in 200 individuals will develop these mulberry-shaped clusters of dilated “low-flow” capillary-venous defects (called cavernomas). Most patients with the sporadic form (80% of cases) display a single lesion by MRI, and can live symptom free. However, familial inherited cases are autosomal dominant and more often exhibit multiple lesions. Most of these cases also develop symptoms. These symptoms can range from headaches to more devastating epilepsy, seizures, neurological deficits caused by cerebral hemorrhages or even hemorrhagic stroke. Development of pharmacological treatments are especially needed for patients with lesions deep in the brain, brain stem or spinal cord, which are inoperable.

Inherited forms have long been linked to LOF mutations 3 key genes: CCM1/KRIT, CCM2/Malcavernin/MGC4607 and CCM3/PDCD10. Mice globally lacking one allele of Ccm1, Ccm2 or Ccm3 suffer early embryonic lethality from vascular defects, but intriguingly do not develop CCM lesions.[35–37] To test the possible “second hit” hypothesis, studies crossed Ccm1 and Ccm2 heterozygotes into DNA repair-deficient mouse lines, such as Msh2−/− or p53−/− sensitized backgrounds.[38, 39] These mice then developed significant CCM lesions, suggesting similar parallel “two hit” mutations may need to occur to precipitate lesion development. These double genetic hits are likely rare events, but they cause local and dramatic expansion of affected cells. This possibility is supported by elegant inducible multi-color fluorescent reporter analysis, where CCMs were shown to arise from clonal expansion of mutant ECs.[40] To circumvent the rare and stochastic nature of these models, and bypass early systemic vascular developmental defects, conditional transgenic mice have been developed that more closely model the sequence of events experienced by patients.[41] These more faithful mouse models provide better suited genetic platforms to test existing CCM drug options (such as fasudil, tempol and vitamin D3). Below, Table 2 lists mouse models developed to investigate key genetic candidates associated with CCMs.

CAPILLARY MALFORMATIONS-ARTERIOVENOUS MALFORMATIONS (CM-AVMS)

Capillary malformation-arteriovenous malformation (CM-AVM) syndrome is another inherited autosomal dominant vascular disorder. CM-AVMs are characterized by enlarged capillaries near the surface of the skin, often appearing as small (1-2cm diameter) red patches on the face, arms or legs. These are often called “port-wine stain” due to their coloring. In addition, in CM-AVMs, there can also be inappropriate arterial and venous connections. Importantly, almost one third of CM-AVM patients present deeper, more serious associated AVMs.[42]

Familial cases of CM-AVM have led to the identification of LOF mutations of the RASA1 gene. RASA1 is a GTPase-activating protein that negatively regulates the small GTP-binding protein Ras. RASA1 mutations have been found in a staggering 50% of CM-AVM patients. Interestingly, only one allele of RASA1 is mutated in these patients, in all cells, however lesions are regionally localized. It was thus hypothesized that a somatic “second hit” RASA1 mutations are required for VM formation, and this was supported by patient lesion data.[43] Consequently, mouse models were developed using either global ablation or conditional ablation of Rasa1[44], or a knock-in of a point mutation of Rasa1 (R780Q).[45, 46] However, while these models demonstrated an essential role of RASA1 in normal blood vessel and lymphatic valve development, they did not recapitulate CM-AVM. It is thought that this is likely due to an unknown “second hit” required in parallel to loss of RASA1 yet to be found.

Interestingly, RASA1 is a known negative regulator of RAS signaling, and RAS family members KRAS, HRAS and NRAS are all expressed in ECs. However, only activating mutations of KRAS display any VMs. The question arises as what role does the RAS pathway play in loss of vascular integrity? RASA1 and Ras vascular studies further illustrate the need to better understand the genetic pathways underlying normal vessel maintenance.

Below, Table 2 lists mouse models developed to investigate key genetic candidates associated with CM-AVMs.

LYMPHATIC MALFORMATIONS (LMS)

Lymphatic malformations (LMs), also known as lymphangiomas, are rare but often debilitating non-malignant vascular anomalies that result from abnormal development of the lymphatic vasculature. LMs are characterized by fluid-filled cystic structures that can be large (macrocystic), or smaller and more pervasive (microcystic). These lesions can develop anywhere in the body, but are most frequently found in the head and neck region. Lesions can excessively grow, both pre- and post-natally, causing significant discomfort. Resection and sclerotherapy are the most common therapeutic approaches. Cellular and molecular mechanisms underlying LMs remain to this day poorly understood. However, it has been shown again that candidate mutations appear localized to lymphatic endothelial cells (LECs) and not adjacent stromal or mesenchymal cell types.

As previously mentioned, RASA1 mutations have been associated in human CM-AVM. However, inducible deletion of Rasa1 in mice resulted requirement for lymphatic development. Defects in lymphatics were characterized by lymphatic vessel hyperplasia and leakage, as well as lethality associated with chylothorax (lymphatic fluid accumulation in the pleural cavity). Similar defects were found in mice in which conditional deletion of Rasa1 was restricted to LECs. These findings suggest a possible partial role for RASA1 in LM etiology and begs the question as whether parallel accompanying mutations in unknown genes are required for disease progression.

Mutations identified in patient LMs have also pointed to activating mutations in the phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene.[47] PIK3CA mutations can cause hyperactivation of the PI3K-AKT-mTOR pathway. One group used high-throughput sequencing to identify somatic activating mutations of PIK3CA in lesions from patients with generalized lymphatic anomaly (GLA), a disease which displays multifocal LMs.[48] Going further, the group demonstrated that the mTOR inhibitor, rapamycin, was capable of preventing lymphatic hyperplasia and would protect mice expressing a constitutively active form of PIK3CA (H1047R). These findings demonstrate the power of a useful mouse model and how it can be used to advance our understanding of a number of key genetic defects, as well as pave the path to therapeutic innovation.

Below, Table 2 lists the mouse models developed to investigate key genetic candidates associated with LMs.

CONCLUSION

As the long tables in this review attest, many different mouse models have been developed over the last few decades to address the origins of VMs, and steady progress is being made in identifying key genes required for vascular stability. However, much remains to be learned. Dissecting the molecular underpinnings of VMs will be critical for the development of novel therapies for patient care. Thankfully, the rise of rapid and relatively inexpensive single cell sequencing has catalyzed a revolution in our ability to identify mutations in VMs, both known and unknown. Mutations are constantly being identified and reported in patient VM lesions as these tissues are excised and analyzed. Genes such as MAP2K1, FBN1, GLMN, EPHRINB2, EPHB4, PTEN, TEK, AKT1, GNA14, IDH1/2, GNA11 and GNAQ have all been repeatedly implicated in VMs in the literature, and sequencing of VM lesions recently reconfirms them as promising candidates.[75] Do they play key roles? Can mouse models mimic their functions in mouse VMs? Do we have the tools to test their roles in the right ECs? Although most current vascular mouse models depend on identified promoters or gene loci which drive expression in all ECs or in wide subsets thereof, recent innovative technologies allow intersectional gene regulation methods, offering promising organ-specific approaches to EC gene regulation (Dre-Cre).[76] These methods allow us, for now, to bypass the need for identification of organ-specific EC regulatory regions and to turn genes on or off at will, where and when we want.

This is indeed an exciting and promising time, as mouse models with either single or compound gene mutations can now be generated relatively easily, for the purpose of evaluating the role of such candidates in vascular integrity and in the etiology of VMs. We just need to do it. Finding molecular pathways responsible for vascular defects is likely to point us towards drugs that might prove effective as treatments, opening the door wide open to personalized therapies.

KEY POINTS.

  • Genetic mouse models can be used to manipulate gene expression in the vasculature in a spatiotemporal manner.

  • Inducible and mosaic deletion of genes in an endothelial cell specific manner allows recapitulation of genetic conditions underlying vascular anomalies.

  • Patient vascular lesion sequencing studies have yielded numerous candidate genes for which no mouse model yet exists.

Acknowledgements

Thank you for the inspiration, Luisa Iruela-Arispe, Salim Seyfried, Miikka Vikkula, Elisabeth Tournier-Lasserve and Josef Penninger. Grateful to Xiaowu Gu and Neha Ahuja for critical reading of the review and comments. Apologies to the many authors whose works were not cited due to space requirements.

Financial support and sponsorship

This work was supported from a grant from the National Institutes of Health (HL126518, HL136139).

REFERENCES AND RECOMMENDED READING

Papers of particular interest have been highlighted as:

• of special interest

•• of outstanding interest

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