Vascular deficiency of Smad4 causes arteriovenous malformations: a mouse model of Hereditary Hemorrhagic Telangiectasia
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that leads to abnormal connections between arteries and veins termed arteriovenous malformations (AVM). Mutations in TGFβ pathway members ALK1, ENG and SMAD4 lead to HHT. However, a Smad4 mouse model of HHT does not currently exist. We aimed to create and characterize a Smad4 endothelial cell (EC)-specific, inducible knockout mouse (Smad4f/f;Cdh5-CreERT2) that could be used to study AVM development in HHT. We found that postnatal ablation of Smad4 caused various vascular defects, including the formation of distinct AVMs in the neonate retina. Our analyses demonstrated that increased EC proliferation and size, altered mural cell coverage and distorted artery–vein gene expression are associated with Smad4 deficiency in the vasculature. Furthermore, we show that depletion of Smad4 leads to decreased Vegfr2 expression, and concurrent loss of endothelial Smad4 and Vegfr2 in vivo leads to AVM enlargement. Our work provides a new model in which to study HHT-associated phenotypes and links the TGFβ and VEGF signaling pathways in AVM pathogenesis.
KeywordsSmad4 Arteriovenous malformations (AVM) Hereditary hemorrhagic telangiectasia (HHT) Vegfr2 TGFβ
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that affects 1 in 5000 people worldwide [1, 2]. HHT patients commonly exhibit: spontaneous, recurring nosebleeds; small lesions on mucous membranes called telangiectasias; and/or larger visceral lesions known as arteriovenous malformations (AVMs) [3, 4]. AVMs, which are direct connections between arteries and veins, are most commonly found in major organs such as the brain, liver or lungs. These lesions present a serious health risk and can lead to decreased quality of life and/or early death due to hemorrhaging, stroke and aneurysms [3, 5, 6, 7, 8].
Approximately 85% of HHT cases are linked to heterozygous loss-of-function mutations in the transforming growth factor beta (TGFβ) cell surface receptors activin receptor-like kinase 1 (ALK1, HHT2) or endoglin (ENG, HHT1) [9, 10]. A small subset of HHT patients (~ 4%) exhibit haploinsufficiency of Mothers against decapentaplegic homolog 4 (SMAD4, JP/HHT) and commonly present with juvenile polyposis syndrome (JP) [11, 12]. SMAD4 is a transcription factor found in nearly all cell types [13, 14], where it serves as the central conduit through which canonical TGFβ signaling proceeds, including ALK1 and ENG signaling . However, despite the key role of SMAD4 in the TGFβ pathway, the mechanisms by which it contributes to HHT pathogenesis remain unknown. In fact, virtually all HHT animal studies have focused on the Alk1 and Eng receptor interface of the TGFβ signaling pathway, whereby endothelial loss of Alk1, or Eng or blockade of the TGFβ pathway via Bmp9/10 ligand-blocking antibodies results in HHT-associated phenotypes [16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26]. What little we know about the in vivo role of SMAD4 in the vasculature comes from embryonic studies. These studies revealed that SMAD4 plays a critical role in blood vessel remodeling and maturation , integrity of the blood-brain barrier endothelium  and regulating coronary artery size . Conversely, nothing is known about SMAD4 function in the postnatal vasculature as homozygous loss of Smad4 is embryonic lethal . Therefore, due to limited information on how SMAD4 contributes to the developing endothelium, it is unclear how SMAD4 defects lead to HHT phenotypes, such as AVM formation.
In order to better understand SMAD4’s contribution to HHT pathogenesis, we created an inducible, endothelial cell (EC)-specific Smad4 knockout mouse model (referred to as Smad4-iECKO). We find that induced deletion of Smad4 leads to various vascular defects including the formation of AVMs. In addition, we show that SMAD4 influences EC proliferation, EC size, mural cell coverage and artery–vein gene expression. Utilizing this new Smad4-iECKO model, we found that deletion of Smad4 leads to decreased levels of vascular endothelial growth factor receptor 2 (VEGFR2) expression. Furthermore, concurrent loss of endothelial Smad4 and Vegfr2 in vivo leads to an increased AVM severity. This work provides a new model for the HHT field and presents evidence that the TGFβ and VEGF pathways may be linked in AVM pathogenesis.
EC-specific deletion of Smad4 causes multiple vascular defects, including AVM formation
To confirm Smad4 deletion in the endothelial lineage, quantitative PCR (qPCR) was performed using RNA from P7 Smad4f/f (control) and Smad4-iECKO isolated retinal ECs which revealed an ~ 80% reduction in Smad4 mRNA transcripts (Fig. 1B). In addition, using a Rosa26-EYFP transgenic reporter line  we confirmed that Cre-recombinase was specifically expressed in blood vessels, while absent in control blood vessels (Fig. S1C–D′). These data demonstrated efficient and specific Smad4 knockdown in the ECs of Smad4-iECKO retinas.
In order to assess the effects of Smad4 depletion on vascular development, Smad4f/f control and Smad4-iECKO P7 retinas were labeled with the vascular marker Isolectin-IB4. We observed numerous arteriovenous malformations (AVMs) in the retinas of Smad4 mutants (Fig. 1C–F), similar to those identified in Alk1- and Eng-deficient mice [17, 18, 20, 24, 33, 34]. Approximately 82% of our Smad4 mutants had AVMs, whereas AVMs were absent in all controls. Multiple AVMs were seen in 52% of Smad4-iECKO mice with an average of 1.732 AVMs per mutant retina (Fig. 1K). AVMs varied in morphology but were easily identifiable because the shunts appeared grossly enlarged in comparison with normal capillaries (Fig. 1C–F and Fig. S2A–H). AVMs were almost always located near the center of the retina, likely due to blood flow patterns in HHT models as previously described . In Smad4-iECKO mice, AVMs form around P5 (data not shown) and either did not form or were smaller if Tx was administered after P1 (Fig. S2I–K).
Loss of Smad4 also caused a noticeable reduction in vascular outgrowth toward the retinal periphery (Fig. 1C, D, L). For this reason and because Alk1 mutant zebrafish exhibit EC migratory defects , we aimed to further assess Smad4 function on EC migration in vitro. We generated stable C166 mouse EC lines that expressed either nonsilencing-shRNAs or Smad4-shRNAs. In comparison with nonsilencing-shRNA, Smad4-shRNA C166 cells showed an approximately 60% reduction in levels of Smad4 transcripts and a diminished capacity to migrate and repopulate wounds in a scratch assay (Fig. S3A–D).
Although outgrowth was stunted, the number of tip cells was not significantly changed in Smad4 mutant retinas (data not shown). Similarly, quantification of vascular densities showed no statistical differences on average (data not shown). This is likely due to the high variability between mutants, as we observed some mutants that displayed significant increases in density in the tip region, while others were indistinguishable from controls (Fig. S4). Although there was variability among vascular density of mutants, one consistent phenotype was that of increased artery and vein diameters (Fig. 1G–J, M, N). Increases in vessel diameter and stunted outgrowth were also even seen in the ~ 20% of mutants that did not express AVMs, suggesting that these events may precede AVM development.
Collectively, these results demonstrate that Smad4 is required for proper vascular growth and vessel morphology in the postnatal retina. Furthermore, the presence of AVMs verifies that loss of Smad4 in mice recapitulates phenotypes associated with HHT patients, thus making this a suitable model for studying Smad4 mechanisms of HHT.
Smad4 depletion causes increases in EC proliferation and size
Defective mural cell coverage in Smad4-iECKO mice
Conversely, AVMs are associated with a reduction in pericyte coverage [20, 35]. Smad4 deficiency has been shown to affect EC–pericyte interactions resulting in loss of pericyte coverage in the developing brain vasculature . To test whether this relationship exists in Smad4-iECKO retinas, we investigated pericyte coverage using an anti-neuron-glial antigen 2 (NG2) antibody. We found that compared to controls, Smad4 mutant retinas exhibit a marked reduction in NG2 protein accumulation in the retinal vasculature (Fig. 4D–E'). Furthermore, qPCR analysis on whole retina samples verified that Ng2 and Desmin (a pericyte marker) mRNA levels are significantly diminished when Smad4 is deleted (Fig. 4F, G). Since the platelet-derived growth factor (PDGF) signaling pathway plays a significant role in recruiting pericytes to blood vessels , we assessed whether changes in expression of the endothelial-secreted PDGFB ligand could account for the loss of pericyte coverage in Smad4 mutants. qPCR results showed that Pdgfb transcript levels are similar in control and Smad4-iECKO whole retinas (Fig. 4H), suggesting that other factors are responsible for the reduced pericyte presence in Smad4-deficient retinas. Overall, our results are consistent with other HHT models in that vSMC coverage inappropriately extends to AVMs and veins, while pericyte coverage is reduced in a Smad4-deficient background.
Artery–vein identity is disrupted in the absence of Smad4
To further confirm changes in AV gene expression, we used immunofluorescent staining and in situ hybridization techniques on Smad4f/f and Smad4-iECKO retinas. At P7, ENDOMUCIN is largely absent from control retinal arteries (Fig. 5B); however, in Smad4 mutants, we observed distinct protein expression in the arteries (Fig. 5C), suggesting that increased Endomucin mRNA levels in iLECs (Fig. 5A) might be due to enhanced expression in arteries. Analysis of Apelin mRNA showed robust, ectopic expression in the retinal veins and capillaries of Smad4 mutants compared to controls, which completely lacked expression in these vessels (Fig. 5D, E). On the other hand, the apelin receptor, Apj, was present in the AVM but showed no noticeable changes in mRNA expression between Smad4 control and mutant retinas consistent with our qPCR results (Fig. 5F, G). Examination of Ephb4 revealed the loss of transcripts in the capillaries and arteries, although Ephb4 mRNA remained in the veins (Fig. 5H, I). Interestingly, even though overall levels of Ephb4 mRNA were reduced, Ephb4 was still noticeably expressed within the AVM and the artery connected in the AVM. Analysis of Notch4 showed no changes in localization of transcripts between Smad4 control and mutant retinas; however, levels of Notch4 appeared markedly higher in Smad4-iECKO retinas (Fig. 5J, K). In all, the whole retina staining results are consistent with the qPCR analysis performed on iLECs. This also revealed the importance of examining both qPCR levels in the whole vasculature as well as localization changes, as some markers are gained in specific vascular beds (Apelin), while others are lost (Ephb4). More so, some markers can increase in levels without changing localization (Notch4), while others remain the same but are expressed in the AVM (Apj). Therefore, we conclude that loss of Smad4 in the endothelium alters AV gene expression and as other groups have suggested may play a contributing role in the formation of AVMs due to disruptions in vessel identity .
In addition, we quantified the transcript levels of Alk1 and Eng in Smad4 control and mutant iLECs, as previous reports have shown that genetic knockdown of one receptor can lead to changes in expression of the other in vivo [17, 20, 24]. Our qPCR results indicate a significant loss in expression of Eng in Smad4-depleted ECs; however, no changes in Alk1 expression were observed (Fig. 5A).
Loss of Vegfr2 enhances Smad4 mutant phenotypes
Interestingly, reciprocal experiments in Vegfr2 null backgrounds showed slightly different results. As previously observed, complete loss of Vegfr2 (Vegfr2f/f-iECKO) in the retina led to severe vascular defects, including an overall reduction in the vasculature with fewer vessels and a lack of definitive capillaries (compare Fig. 7A, G) . Loss of a single allele of Smad4 in the Vegfr2 null background (Smad4f/+;Vegfr2f/f-iECKO) resulted in similar phenotypes, suggesting that heterozygous loss of Smad4 had little affect on the overall vascular phenotype (Fig. 7H). In addition, combinatorial deletion of Smad4 and Vegfr2 (Smad4f/f;Vegfr2f/f-iECKO) led to AVM formation, but these retinas exhibited very little vascular coverage (Fig. 7I). This suggests that there is a threshold for which loss of Vegfr2 becomes dominant to Smad4 deletion, as was previously reported in Alk1 mutants . In conclusion, our results indicate that reduction in VEGF signaling may contribute to heightened HHT phenotypes during developmental angiogenesis.
Our studies are the first to report a Smad4 animal model of HHT (Smad4-iECKO). We showed that endothelial loss of Smad4 recapitulates vascular phenotypes seen in other HHT mouse models, particularly AVM formation. To better understand Smad4’s role in HHT pathogenesis, we performed a comprehensive characterization of Smad4-iECKO mice. Our results demonstrated that increased EC proliferation and size, alterations in mural cell coverage and disruption in AV gene expression are associated with Smad4-deficient blood vessels. We also provided evidence that loss of SMAD4 causes decreased VEGFR2 expression, and that loss of a single allele of Vegfr2 in the Smad4 null background leads to an increased severity of AVMs.
Comparison of HHT mouse models
Percentage of mutants with AVMs
No reduction 
Smooth muscle coverage
Decreased (only in capillaries) 
No change ; not expressed in AVM
No change (Expressed in AVM) 
No change; expressed in AVM
Eph b 4
No change (Expressed in AVM) 
Reduced expressed in AVM
No change 
Altered VEGFA-induced kinetics 
24–48 h post-Tx Inj 
168–192 h post-Tx Inj
Nonetheless, our work demonstrated that expression of NOTCH signaling components, which are associated with arterial identity, as well as genes connected to venous and tip cell identity, are disrupted in the absence of Smad4 (Fig. 4). We also revealed that these changes can occur in arteries, veins and/or capillaries; however, it is important to note that the AVMs themselves expressed all genes examined regardless of whether the marker was up- or downregulated in other vessel types. When comparing these results to those obtained in Alk1 and Eng mouse models, we noted variations in AV gene expression between all three mutant backgrounds [17, 20, 24, 34, 38]. These differences could be due to tissue-specific effects related to the source tissues examined and/or the vascular expression patterns of Alk1, Eng and Smad4. For instance, some studies examined gene expression in isolated lung ECs , while others utilized brain and/or retinal ECs [20, 34]. Additionally, it is possible that expression levels in various vessel types play a role, as Alk1 is highly expressed in arterial ECs , while Eng is only moderately expressed in arteries . Eng also is expressed highly in capillaries and weakly in veins . In comparison, Smad4 is present in virtually all tissues [13, 14]. However, despite these differences, it is clear that overall disruptions in AV gene expression are consistent between all three mouse models of HHT. Further examination is needed to address whether alterations in AV identity are a primary cause or secondary effect of AVM formation. To this point, our work does not address whether the observed phenotypes and molecular changes are a cause or an effect of AVM formation, as experiments were performed after AVMs developed. This cause/effect relationship has not been explored in Alk1 and Eng models of HHT either. Therefore, future studies addressing this issue will be important for identifying the underlying molecular defects that drive AVM pathogenesis versus those that are secondary effects of AVM formation.
It is also important to note that tamoxifen-inducible murine models of HHT have several limitations. HHT phenotypes arise in patients due to mutations (most commonly missense mutations) that lead to haploinsufficiency . In contrast, mouse models of HHT often utilize null genetic backgrounds because loss of one allele of Alk1, Eng or Smad4 does not result in consistent presence of AVMs in predictable locations [21, 26, 53, 54, 55]. Furthermore, HHT patients harbor germline mutations, which manifest from gestation and remain throughout adulthood. However, in mice, complete loss of Alk1, Eng or Smad4 during gestation results in embryonic lethality making it impossible to study their postnatal impact on HHT [16, 27, 33, 56, 57]. For this reason, the mouse retina has become an effective model to study AVM formation; the retinal vasculature forms directly after birth allowing researchers to assess developmental angiogenesis, similar to vessel growth that would be seen in a developing human. Although these models do not perfectly mimic the genetic background of HHT patients, retinal AVMs form at consistent rates and locations providing a reliable model to investigate the mechanisms of AVM formation.
In our Smad4-iECKO retinas we noted delayed angiogenic outgrowth similar to Eng mutants [17, 34], while Alk1 mutant retinas did not exhibit reduction in vascular outgrowth . Interestingly, our Smad4-iECKO mice exhibit a significant reduction in Eng transcript levels but show no changes in Alk1 mRNA levels (Fig. 5A). This could account for the observed similarities in reduced vascular outgrowth between Smad4 and Eng, but not Alk1 mice. However, this result also illustrates the complex association between the TGFβ pathway and HHT, as Eng expression levels are reduced in the Alk1 mouse models of HHT [20, 24], yet show no changes in outgrowth. To our knowledge, it is unknown what happens to levels of Alk1 expression in the Eng HHT model, or whether Smad4 levels are affected in either Alk1 or Eng mouse models. Moving forward, it will be important to understand the association between Alk1, Eng and Smad4 in HHT because even though it is expected that all three cooperate in a linear manner in the TGFβ pathway, differences in phenotypes (Table 1) suggest this might not be the case.
The overall objective of our work was to develop a Smad4 model of HHT that could be used to identify the TGFβ targets that drive AVM formation, as almost nothing is known about these downstream effectors. To this end, we explored a possible link with the vascular endothelial growth factor (VEGF) signaling pathway that has been previously suggested in other HHT models [21, 24, 26, 34, 42, 53]. For instance, homozygous-induced deletion of Alk1 or Eng in adult mice requires the presence of exogenous VEGF before AVMs will form in the brain, suggesting that activation of the VEGF pathway is needed for AVM formation [21, 26]. To this end, VEGF neutralizing antibodies have been shown to prevent wound-induced skin AVMs from developing in Alk1-deficient mice . Furthermore, in the absence of Alk1 and Eng, several studies have reported increased Vegfr2 expression and altered VEGFR2 kinetics in vitro [24, 34, 42]. In contrast, our data showed that loss of Smad4 led to a reliable and significant decrease in Vegfr2 expression both in vitro and in vivo (Fig. 6). This is consistent with a previous study on human patients with cerebral brain AVMs where there was a marked decrease in Vegfr2 expression . Contrary to other HHT studies, the reduction of Vegfr2 in Smad4-iECKO mice could potentially be attributed to the downregulation in Nrp1, a VEGFR2 co-receptor. Studies have shown that decreased Nrp1 levels correlate with reduced Vegfr2 expression [59, 60]. Although other HHT studies did not find reduced Vegfr2 levels, homozygous deletion of both Smad4 and Vegfr2 produced similar results to those obtained in double Alk1- and Vegfr2-deficient retinas . In each study, deletion of both alleles of Vegfr2 in the Alk1 or Smad4 null backgrounds resulted in inhibition of retinal vascular development, suggesting that appreciable loss of Vegfr2 in the absence of either Alk1 or Smad4 overrides HHT-like phenotypes because the vasculature is severely underdeveloped (Fig. 7). We did note that AVMs still formed in both experiments at fewer and similar rates in Alk1 and Smad4 mutants, respectively. However, in further studies we demonstrated that loss of a single Vegfr2 allele in the Smad4 mutant background led to an enhancement of vascular phenotypes associated with Smad4-iECKO retinas; the vascular front exhibited a consistent increase in density and AVMs showed a substantial enlargement. Alternatively, increased AVM size could be attributed to altered blood flow rates, hemodynamics forces and/or rates of oxygen diffusion caused by the overall stunted growth of the mutant blood vessels, rather than due to the loss of VEGFR2 directly. Future studies will be needed to understand how these processes are altered in TGFβ mutant backgrounds and how those contributions may affect severity of AVMs.
This SMAD4-VEGFR2 association is somewhat contrary to the clinical use of bevacizumab (also known as Avastin), which is a humanized anti-VEGF monoclonal antibody that sequesters VEGF to prevent it from binding both VEGFR1 and VEGFR2 subsequently hindering angiogenesis [61, 62]. Bevacizumab is currently used as a palliative therapy for HHT where it alleviates symptoms such as chronic nosebleeds but is not considered a long-term therapy . Studies on the use of bevacizumab have been performed in mature vascular networks, namely that of adult humans and mice [64, 65]. Little information is known about the effects of bevacizumab in children or developing/remodeling vascular networks. Our work suggests that the connection between SMAD4 and VEGFR2 is different during developmental angiogenesis, when AVMs are thought to form, as compared to mature, established vascular networks. Therefore, further research on the effects of bevacizumab in developing vascular networks is needed, as our results indicate that bevacizumab may enhance developmental HHT phenotypes.
Materials and methods
All animal experiments were performed in accordance with Tulane University’s Institutional Animal Care and Use Committee policy. To create our Smad4-iECKO mouse model, we crossed an endothelial-specific, tamoxifen-inducible Cre-driver line (Tg(Cdh5-CreERT2)1Rha, further referred to as Cdh5-CreERT2)  with a conditional Smad4 mouse (Smad4f/f) . To confirm that Smad4 was being knocked out only in ECs, we mated Smad4-iECKO mice with a Rosa26-EYFP reporter mouse (Gt(ROSA)26Sortm1(EYFP)Cos) . Induction of tamoxifen was done using 0.075 mg tamoxifen (Sigma T5648) per gram of body weight on postnatal days 1 and 4. Note: For Vegfr2-iECKO mice only one injection of Tx was given on P1. For experiments, Smad4f/f;Cdh5-CreERT2 (otherwise referred to as Smad4-iECKO) mice were the experimental group, while Smad4f/f littermates were used as controls. Genotyping primers and conditions can be found in supplemental methods.
Hematoxylin and eosin staining of murine lungs
Neonatal lungs were dissected from postnatal day 8 pups and fixed for 4 h in 4% PFA at 4 °C. The lungs were then embedded in paraffin and sectioned at 10 µm. Sections were washed in xylenes twice then put through a rehydration series. Slides were placed into hematoxylin solution for ~ 1 min and then rinsed with water for several minutes. This process was repeated for eosin staining. Slides were then mounted with Permount (Thermo).
Retinal whole mount stains
Retinas were dissected and stained as previously described . The following antibodies were used at a 1:100 concentration: αSMA (Sigma C6198), cleaved CASPASE-3 (Cell signaling 9661), COLLAGEN IV (Millipore AB756P), ENDOMUCIN (Santa Cruz 6415), ERG (Abcam 92513), GFP (Aves GFP-1020), KI67 (Cell Signaling 9449), NG2 (Millipore 5320), PECAM/CD31 (BD 553370), VEGFR2 (BD 555307). Additionally, the following immunofluorescent stains were performed according to the manufacturer’s instructions: Dapi (Life Technologies R37606), Isolectin-488 (Invitrogen 21411), Isolectin-594 (Invitrogen 21413), Isolectin-647 (Invitrogen 32450). Confocal images were taken at the same exposure settings for both mutant and control retinas, so fluorescent intensity could be compared.
In situ hybridizations
In situ hybridizations were performed as previously described . In situ hybridizations were performed in batches where mutants and controls were subjected to the colorimetric reaction for the same period of time so that results could be compared. The following probes were synthesized from plasmids containing: Apelin (Dharmacon), Apj (Dharmacon), Ephb4, Notch4. Images were taken using a Leica M205 FA stereomicroscope.
Isolation of endothelial cells
Retinal and lung endothelial cell isolation were performed as previously described . Briefly, tissue (either lung or retina) was digested in a collagenase/dispase solution and minced into fine pieces. After obtaining a single cell suspension, sheep anti-rat IgG dynabeads (Invitrogen 11035) coated with PECAM/CD31 antibody (BD 553370) were used to isolate endothelial cells. Cells were either used for RNA collection immediately or allowed to grow in EGM-2 medium for one week before being used for protein or RNA collection.
qPCR and analysis
All quantitative real-time PCR (qPCR) experiments were performed using RNA isolated with a GeneJET RNA Purification Kit (Thermo K0732) and quantified using a Nanodrop (Thermo). For each sample, 500–1000 ng of RNA was used for cDNA synthesis using a iScript cDNA Synthesis Kit (Bio-Rad). qPCRs were run using SYBR green mastermix (Thermo K0221) on a Bio-Rad CFX96 Touch Real-Time PCR Detection machine. Analysis was performed using the double delta Ct method, and statistics were generated using GraphPad Prism. For all qPCR experiments, three independent biological replicates were used and three technical replicates were performed per sample. Primers were verified for specificity and efficiency and can be found in supplemental materials.
Quantification of retinal images
All retina images were analyzed using Nikon NIS-Elements AR Analysis 64-bit software, and ImageJ software was used to measure vascular outgrowth, cell area and cell density.
GraphPad Prism software was used for all statistical analysis. For all statistics, sample size (n) indicates the number of independent biological samples. A minimum of three technical replicates was included per sample. For statistical analysis, we ran unpaired two-tailed Student’s t test where a p value of < 0.05 was considered significant.
We would like to thank Masanori Hirashima and Ralf Adams for the use of the Vegfr2-floxed and Cdh5-CreERT2 mice, respectively. The Notch4 and EphB4 in situ plasmids were supplied from Ondine Cleaver. This work was supported by start-up funds from Tulane University, funds from the Tulane Committee of Research, and the Department of Defense Investigator-Initiated Research Award (PRMRP160198) [S.M.M.]. This work was supported by funds from the Louisiana Board of Reagents [A.M.C.]. We would also like to acknowledge Harvard Primer Bank and NCBI Primer-Blast for primers used in qPCRs.
Compliance with ethical standards
Conflict of interest
We have no competing financial interests.
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