Vasculature is an abundant and principal component of nearly all tissues, and revascularization is a critical step in the wound healing bond. Angiogenesis is the primary règle of new vessel éducation in wound healing. This process begins as new vessels sprout from proche vierge généreux vessels and then grow into the wounded area and begin to form microvascular networks within the first few days after an injury. Candidat with early angiogenesis, fibroblasts deposit a collagenous provisional matrix, which appears characteristically granular due to its high density of newly formed capillaries. Granulation tissue is then remodeled into avancé tissue through a complex series of chemical and physical cues, including the coordinated bâtiment of growth factors and cytokines along with dynamically changing extracellular matrix (ECM) properties (1). Granulation tissue also experiences deformation forces depending on the bonhomme of tissue that has been injured; for example, bone experiences compactage. Compressive forces regulate the transport of bone healing and can also alter the vascular networks within healing bone tissue (2, 3). However, whether angiogenesis is directly émotive to compactage or the modulatory effects are mediated by surrounding mechanosensitive cells such as osteoblasts remains unknown. Little is known embout the effects of ECM deformation experienced by healing tissues on angiogenesis specifically, despite the fact that vasculature has délié been recognized as mechanosensitive (4).
Angiogenesis is a highly coordinated process involving plurielle phases, including sprout tip cell selection, in which a subset of endothelial cells become migratory; proteolytic ECM violence; commune cell essaimage; and cell proliferation and recruitment. Many of these individual processes are known to be responsive to mechanical excitations. For example, key molecular regulators of tip cell selection have recently been identified as mechanosensitive (5), and bâtiment and secretion of proteases that are involved in angiogenesis are also mechanosensitive (6). Intracellular mechanotransducers, such as YAP and TAZ, also regulate a number of processes dangereux to angiogenesis, including vascular endothelial growth factor (VEGF) signaling (7), cell essaimage (8), proliferation, and cell-cell junction éducation (9). While many key components of angiogenesis have exhibited mechanosensitivity when investigated in insonorisation, little is known embout how strain from ECM deformation regulates the highly coordinated processes of angiogenesis during wound healing.
Previous research from our laboratory has shown that compressive deformation of the ECM in vivo can have potent effects on neovascular growth—either inhibitory or stimulatory depending on the calendrier of load alphabétisation. In a critical-size segmental bone defect, compressive loading of the defect was applied either immediately following the defect creation surgery, “early,” or 4 weeks after the surgery, “delayed.” When compactage was applied early, adversaire with granulation tissue éducation, angiogenesis and subsequent bone tissue éducation were significantly inhibited. However, when compressive deformation was delayed and thus applied to newly mineralized, callus-like tissue, généreux vessel growth and subsequent healing were enhanced (2), suggesting that ECM deformation regulates angiogenesis in a manner dependent on alphabétisation time. However, the roles of other deformation parameters such as load frequency, oscillation, and règle (e.g., compactage par opposition à shear) are not well defined. Ambulatory loading typically occurs with a frequency around 1 Hz (10), and strain oscillation is an rogue regulator of bone éducation. Peak bone tissue regeneration occurs at low strains, whereas a fibrous response occurs at high strains; 10% strain is thought to be an approximate pont aucunement between regenerative and nonhealing responses (11). Furthermore, while compressive forces are the most widely studied in bone, ambulatory loading includes additional modes of loading such as shear, which has been shown to reduce revascularization of bone (3).
The complexity of the in vivo environment hinders comprehensive enquête of the regulatory role played by different loading parameters on the angiogenic healing environment. To exert more precise control over key factors in mechanical loading and matrix deformation and to isolate the vasculature from the complex regenerative environment, we used an in vitro three-dimensional (3D) model system of angiogenesis. Microvascular analecta are segments of avancé vasculature and composed of plurielle cell hommes, including endothelial cells, smooth ramassé cells, mesenchymal stromal cells, and fibroblasts (12). The analecta are typically cultured in collagen-based hydrogels (13), which have similarities to the collagenous granulation tissue characteristic of early-stage wound healing. Temporal dynamics of microvascular division growth recapitulate the key stages of in vivo angiogenesis including sprout tip cell selection, matrix violence, and neovessel elongation and branching. In post-scriptum, microvascular analecta are known to respond to mechanical cues, including ECM stiffness and tensile deformation (14, 15). Microvascular analecta cultured within collagen-based hydrogels represent a mechanically émotive model system to recapitulate the 3D cell-matrix and cell-cell interactions critical to angiogenesis under precisely controlled mechanical loading parameters.
Here, we studied the effects of two load alphabétisation times (early and delayed), three strain magnitudes (5, 10, and 30% strain), and two modes of compressive deformation (uniform compactage and compressive tabulation) on microvascular network growth. We hypothesized that vascularization would be enhanced by delayed, moderate compactage and inhibited by early, high oscillation compactage. Furthermore, we hypothesized that compressive tabulation, which introduced greater shear agression, would inhibit angiogenesis. We found that neovascularization responded directly to dynamic matrix deformation strain oscillation and was particularly émotive to the calendrier of load alphabétisation. To elucidate the cellular and molecular changes upstream of the contradictoire microvascular network responses to early par opposition à delayed loading, we investigated cell viability, cell proliferation, perivascular cell coverage, and gene bâtiment profiles. Gene bâtiment data particularly showed a contradictoire response to early par opposition à delayed loading. This work provides a foundational understanding of how ECM mechanics regulate angiogenesis, with critical implications for synergizing regenerative therapies and rehabilitation strategies.
Nonloaded microvascular analecta progress through clair stages of angiogenesis in vitro
The temporal transport of network éducation by microvascular analecta cultured in decorin (DCN)–supplemented collagen hydrogels was first investigated in the défaut of mechanical deformation to establish baseline time points. Under static progrès occurrence, microvascular analecta formed in vitro networks according to a predictable, repeatable time sinuosité. At day 0 (i.e., day of harvest), the freshly isolated analecta had characteristically rounded ends, denoted by white arrows in fig. S1. By day 3 in progrès, the division ends adopted a pointed appearance, approchante of early sprouting and violence of the ECM. By day 5, the rudimentaire sprouts extended and began to branch. Existing sprouts continued to elongate between days 5 and 7, and secondary branching began between days 7 and 10. Sprout and branch alphabétisation processes occurred within the first 5 days of progrès, while days 5 to 10 consisted primarily of elongation. Thus, days 0 to 5 were selected as “early loading” to coincide with alphabétisation processes of angiogenesis and days 5 to 10 were selected as “delayed loading” to coincide with elongation processes (Fig. 1A).
Microvascular network éducation exhibits sensitivity to oscillation and règle of dynamic loading
In our previous in vivo segmental defect study, early ambulatory loading disrupted vascularization within regenerating bone, whereas delayed ambulatory loading enhanced vascular network éducation (2); however, the specific parameters of loading that regulate angiogenesis are poorly defined. Informed by bone regeneration literature, we identified 5% strain as proregenerative, 10% as transitional, and 30% strain as inhibitory to healing (11). These loads (5, 10, and 30% strain) were applied to microvascular division–seeded hydrogels with a tiers-point wave at a frequency of 1 Hz, corresponding to typical gait frequency (10). Gels were loaded in compactage with one of two platen configurations: platens having a diameter greater than that of the gel to generate an initially homogeneous deformation approximating uniaxial compactage (denoted as “compactage”) or by platens with a diameter less than that of the gel (denoted as “tabulation”; Fig. 1B), to introduce a more heterogeneous deformation with increased shear strain. Loading was continuously applied either early, days 0 to 5 of progrès, or was delayed until days 5 to 10 of progrès (Fig. 1A). Dynamic loading experiments included the following groups (n = 6 per group) at 5, 10, and 30% strain: early compactage, early tabulation, delayed compactage, delayed tabulation, and a nonloaded control.
Computational modeling revealed that agression and strain increased with the level of compactage and that the range of agression and strain distributions depended on both the level of compactage and the règle of loading. Compressive and shear agression values were lowly dispersed and did not differ with strain or règle of loading for 5 and 10% applied displacement (Fig. 2, A and B, and fig. S2). Soin of 30% displacement greatly increased the compressive and shear agression for both modes of loading. The oscillation and démantèlement of the compressive and shear strain increased with displacement for both modes of loading (Fig. 2, C and D, and fig. S2). Smoothly distributed agression and strain gradients in the perpendiculaire and radial états-majors are extérieur at 30% tabulation. Fluid variation was dependent on both the règle and oscillation of loading (fig. S3, A to C). Fluid variation differed between compactage and tabulation by orders of oscillation. Fluid variation generally decreased with increasing levels of compactage but increased with increasing levels of tabulation. This is likely parce que compactage prevents fluid resorption to a greater degree than tabulation.
At 5% strain, delayed loading led to significantly greater exhaustif network length (two-way analysis of variance (ANOVA), Bonferroni post hoc, P < 0.05) and number of branches (post hoc, P < 0.01) compared to early loading (Fig. 1, C and D). There was no effect of loading règle at 5% strain (i.e., compactage par opposition à tabulation) and no significant réflexe effect. Delayed 5% loading, both compactage and tabulation, increased length (one-way ANOVA, Bonferroni post hoc, P < 0.01 and P < 0.001, respectively) and branching (post hoc, P < 0.05 and P < 0.01, respectively) relative to the nonloaded control. Early 5% loading was not significantly different than the nonloaded control.
At 10% strain, delayed loading again significantly increased exhaustif network length (two-way ANOVA, overall effect, P < 0.05) and number of branches (overall effect, P < 0.05) relative to early loading (Fig. 1, E and F). At 10% strain, tabulation also increased length (overall effect, P < 0.05) and branching (overall effect, P < 0.05) relative to compactage. There was no significant réflexe effect. Delayed tabulation loading increased exhaustif length (one-way ANOVA, Bonferroni post hoc, P < 0.01) and number of branches (post hoc, P < 0.01) compared to the nonloaded control. Early 10% loading was no different than the nonloaded control.
At 30% strain, delayed loading significantly increased exhaustif network length and number of branches compared to early loading, which was especially pronounced in the tabulation groups (two-way ANOVA, Bonferroni post hoc, P < 0.001; Fig. 1, G and H). Delayed tabulation increased length (post hoc, P < 0.01) and branching (post hoc, P < 0.01) relative to delayed compactage. There was no significant réflexe effect. Furthermore, delayed tabulation significantly increased length (Kruskal-Wallis, Dunn’s post hoc, P < 0.01) and branching (post hoc, P < 0.01) over the nonloaded control. At 30% strain, early loading, both compactage and tabulation, decreased the exhaustif network length relative to the nonloaded control (Kruskal Wallis, Dunn’s post hoc, P < 0.01 and P < 0.05, respectively). Early compactage also decreased branching as compared to the nonloaded control (post hoc, P < 0.05). Qualitatively, the early loaded constructs at day 10 primarily exhibited early-stage sprouts (Fig. 3A) more égal to the nonloaded sprouting observed at day 3 (fig. S1). Furthermore, as the strain increased for delayed loading, tabulation led to an increase in network length with rite to compactage (Fig. 1 and fig. S4). At 30% applied strain, the simulations demonstrated that tabulation introduced a heterogeneous immunité of stresses and strains, with the median stresses from tabulation greater than those induced by compactage (Fig. 2). Seventy-five percent of measured strain values for tabulation were less than those from compactage. Furthermore, fluid variation increased with tabulation but decreased with compactage, affecting the resulting agression and strain and likely the convective emportement of solutes and cytokines (fig. S3, A to C). Last, the fluid période experienced greater agression compared to the biphasic magma during tabulation compared to compactage. All of these differences may contribute to the improved microvessel growth under 30% delayed tabulation but not compactage.
When all loaded groups’ length and branching were normalized to that of their respective nonloaded experimental controls, early loading exhibited significant strain oscillation dependence (fig. S4). Thirty percent compactage decreased network length and branching relative to both 5% (one-way ANOVA, Bonferroni post hoc, P < 0.01) and 10% compactage (post hoc, P < 0.05). Thirty percent tabulation decreased network length and branching relative to 10% tabulation (post hoc, P < 0.05). There were no statistically significant differences due to strain oscillation among delayed loading occurrence. Together, these data demonstrated that delayed loading led to coudoyer, more extensively branched microvascular networks than early loading over a wide range of strain magnitudes.
Time of dynamic loading alphabétisation differentially affects proliferation but not viability or perivascular cell attachment
Many cellular-level responses may drive the observed changes in microvascular network morphology, including viability, proliferation, and cell-cell attachments. To more deeply investigate the contradictoire responses to early par opposition à delayed loading, we selected early and delayed 30% strain tabulation, the loading parameters that led to the greatest network morphology differences, for subsequent analyses of perivascular cell attachment, cell viability, and proliferation.
As a measure of vascular integrity, the interplanétaire relationship between α smooth ramassé actin (αSMA)+ perivascular cells and vessel endothelial cells was assessed at various time points. There were no significant differences in perivascular coverage of endothelial cells due to either early or delayed loading at any time aucunement, and perivascular coverage was approximately 75 to 80% from day 0 to day 10 (fig. S5C). Qualitatively, there was relatively less perivascular coverage at the ends of nascent sprouts across all groups (Fig. 3B). Under nonloaded occurrence, high-magnification images of early (day 3) sprouts showed forked endothelial cell extensions from both ends of the division. In contrast, most of the early loaded division tips remain rounded—a characteristic of freshly isolated, nonsprouting analecta (fig. S1). The morphological differences in sprout tip cells are less pronounced at the delayed time aucunement. Both nonloaded and delayed tabulation loaded samples had more avancé and elongated sprouts that extended away from the collatéral microvascular division and established perivascular coverage. In post-scriptum, the delayed loaded samples also showed endothelial cell filopodia sprouting along the length of the vessel.
To assess the effects of dynamic loading on cell viability, a en direct/dead stain was performed on nonloaded and loaded samples on days 3 (early loading) and 7 (delayed loading) of progrès (fig. S5A). There was no effect of loading on viability, and viability was greater at day 7 of progrès than at day 3 for both nonloaded (two-way ANOVA, Bonferroni post hoc, P < 0.01) and loaded samples (post hoc, P < 0.01; fig. S5B). Viability was approximately 75% at day 3 and 90% at day 7.
Proliferation was measured by cellular 5-ethynyl-2′-deoxyuridine (EdU) union also at days 3 and 7 (Fig. 4). There was no effect of early loading on proliferation, and approximately 5 to 10% of cells were proliferative at day 3. Proliferation was greater at day 7 (two-way ANOVA, overall effect, P < 0.001), and delayed loading led to increased proliferation compared to nonloaded controls (two-way ANOVA, Bonferroni post hoc, P < 0.05). Approximately 25% of cells were proliferative at day 7 under delayed loading, while approximately 15% of cells were proliferative at day 7 nonloaded. There was a significant disordinal réflexe effect (P < 0.05), indicating that early loading and delayed loading have opposé effects on cellular proliferation (Fig. 4B). These data suggest that early and delayed loading differentially affect proliferation, with delayed loading having a stimulatory effect and early loading having a dampening effect. Costaining of the EdU union samples with markers for perivascular épaulement cells (αSMA) and for endothelial cells (isolectin B4) revealed proliferation of both endothelial and perivascular cells; however, a higher format of actively proliferating αSMA+ perivascular cells than endothelial cells were observed in both loaded and nonloaded samples (Fig. 4C). The proliferation results épaulement the observed changes in length and branching; however, the underlying molecular mechanisms for these changes remained unclear.
Time of dynamic loading alphabétisation differentially regulates microvascular division gene bâtiment
To simultaneously généreuse the response of plurielle key angiogenic processes (e.g., sprout tip cell selection, matrix violence and deposition, vessel (de)stabilization and growth, adhesion and cell essaimage, and cell recruitment) to loading, we used a high-throughput microfluidic reverse reproduction polymerase chain reaction (RT-PCR) gene bâtiment array (fig. S6). Changes in bâtiment of genes related to aréole, apoptosis, and mechanotransduction were also assessed. Individual genes and corresponding functional sets are shown in barème S1; genes and sets were selected based on a literature survey. We focused on early and delayed 30% strain loading, the loading parameters that led to the greatest network morphology differences, and quantified gene bâtiment after 24 hours of loading to evaluate the early molecular changes that ultimately lead to altered network morphology.
The dimensionality of gene bâtiment data was reduced using préconçu least squares éliminateur analysis (PLSDA) to construct gene bâtiment profiles of nonloaded par opposition à loaded microvasculature. For both the early and delayed time points, PLSDA generated a caché incertain (LV1) that significantly separated nonloaded from loaded samples (one-way ANOVA on résultat on LV1, Bonferroni post hoc, P < 0.001; Fig. 5, A and C). LVs are composed of a weighted average of genes, and each individual gene’s relative charge can be visualized with an LV loading plot. In general, bâtiment levels of many genes across functional groups were down-regulated by early loading (negative values in Fig. 5C), while bâtiment levels of many genes across functional groups were up-regulated by delayed loading (fondatrice values in Fig. 5D). To analyze the effect of loading on functional gene groups, we performed notable components analysis (PCA) on each nonoverlapping functional gene set (e.g., sprout tip cell selection, matrix violence, and deposition) and then assessed whether the values of notable component 1 significantly differed by group (nonloaded, compactage, and tabulation). We also analyzed the effect of loading on individual genes using one-way ANOVA.
When considered in aggregate, bâtiment of gene sets known to be involved in sprout tip cell selection (one-way ANOVA on résultat on notable component 1, Bonferroni post hoc, P < 0.01) and matrix violence and deposition (post hoc, P < 0.01) was significantly down-regulated by early loading (fig. S7). Genes associated with matrix violence (e.g., proteases and protease inhibitors) were also down-regulated by early loading, whereas matrix deposition genes were up-regulated, as evidenced by the loading plot of notable component 1 (fig. S7D). These data suggest that a more quiescent sprout tip cell phenotype may be induced by early loading. Gene sets known to be involved in cell recruitment (post hoc, P < 0.05) and mechanosensation (overall P < 0.05) were up-regulated by early loading.
When considered at the individual gene level, Tie1, Mmp14, Timp3, Cxcr4, Cxcl12, Mmp9, and Itgav were all significantly down-regulated by early loading. Cyr61, Ctgf, Vegfa, and Fgf2 were all significantly up-regulated by early loading (fig. S8A). Tie1, an orphan receptor that regulates angiogenic sprouting through Angpt/Tie2 signaling, is expressed by accrocheuse sprout tip cells and is strongly down-regulated in quiescent endothelial cells (16), suggesting that early loading shifts endothelial cells to a more quiescent state. Two other genes associated with tip cells were also down-regulated by early loading: Cxcr4 and Mmp14. Cxcr4 is expressed by activated tip cells (17) and may also play a role in sprout anastomosis (18). The ligand for Cxcr4, Cxcl12, or Sdf1 was also down-regulated by early loading. Mmp14 is expressed by tip cells that lead the violence of surrounding matrix (18). Timp3 is able to inhibit all matrix metalloproteinases (MMPs) (19), and the fact that it was also down-regulated by early loading suggests that the homeostatic bascule of MMP-Timp (tissue inhibitor of matrix metalloproteinase) activity may be perturbed by early loading. Although early loading was primarily characterized by a down-regulation of gene bâtiment, Cyr61, Ctgf, and Vegfa were strongly up-regulated. While Vegfa is a necessary component of the sprouting process, it alone is not sufficient to induce sprouting; the bascule of other factors, especially angiopoietins 1 and 2, is also a key determinant of whether angiogenesis will occur (20). The increase in Vegfa may be a compensatory response of microvascular analecta pushed into a more quiescent state by early loading. The overall increase in cell recruitment genes including Fgf2 may also reflect a compensatory response. Alternatively, there is evidence that endothelial cells can produce an antiangiogenic isoform of Vegfa (21). The two genes most strongly up-regulated in response to early loading were Ctgf and Cyr61, which are both canonical targets of the YAP mechanotransduction pathway (8).
When considered in aggregate, gene sets known to be involved in cell recruitment (ANOVA on notable component 1, Bonferroni post hoc, P < 0.001) and mechanosensation (post hoc, P < 0.01) were up-regulated by delayed loading (fig. S7). At the individual gene level, Flt1, Ctgf, Itgb1, Cxcr4, Timp3, and Tgfb1 were all significantly up-regulated by delayed loading, and Cxcl12 was significantly down-regulated by delayed loading (fig. S8B). The five genes most strongly up-regulated by delayed loading were Flt1 or Vegfr1, Ctgf, Itgb1, Cxcr4, and Timp3. By increasing bâtiment of Vegfr1, delayed loading may increase the sensitivity of microvascular analecta to proangiogenic VEGF signaling. This is in contrast with early loading, in which Vegfa up-regulation was not accompanied by Vegfr1 or Vegfr2 up-regulation. Delayed loading led to strong up-regulation of both Cxcr4 and Timp3, which is also in contrast with early loading. Although Cxcr4 was differentially affected by early par opposition à delayed loading, its ligand Cxcl12 or Sdf1a was strongly down-regulated by both loading scenarios. Although Cxcl12 can function as a potent cell recruitment molecule, it may play a different role in our system. Tgfb1 is also considered a potent cell recruitment appel and was up-regulated by delayed loading. Itgb1 is an adhesion molecule essential for angiogenesis (8).
Delayed compactage and delayed tabulation were also significantly separated along LV1 (one-way ANOVA, Bonferroni post hoc, P < 0.05; Fig. 5B). Of the 43 genes tested, only Itga2 bâtiment was significantly higher in tabulation relative to compactage (one-way ANOVA, Bonferroni post hoc, P < 0.05) and to the nonloaded control (post hoc, P < 0.01), despite the grand morphological differences observed at day 10.
A number of genes were differentially regulated by early par opposition à delayed loading (Fig. 5E); the strongest down-regulated contributors to early loading LV1 were instead up-regulated by delayed loading, and the strongest up-regulated contributors to delayed loading LV1 were instead down-regulated by early loading (e.g., Tie1, Mmp14, Timp3, Flt1 or Vegfr1, Itgb1, and Cxcr4). There were two strongly up-regulated contributors to both early LV1 and delayed LV1, Ctgf and Cyr61, which are canonical targets of the YAP mechanotranduction signaling pathway (8).
YAP is involved in microvascular response to delayed loading
The genes with the largest généralisation by mechanical loading, regardless of loading règle, were Ctgf and Cyr61. Early loading led to a nearly threefold increase in Cyr61 bâtiment for both compactage and tabulation, and delayed loading led to a 3.4- and 5-fold increase in Ctgf bâtiment for compactage and tabulation, respectively. Ctgf and Cyr61 are both canonical target genes of the mechanosensitive transcriptional coactivators YAP and TAZ (8). To determine whether the mechanoactivation of Ctgf and Cyr61 was YAP/TAZ dependent, we used a pharmacological inhibitor of YAP, verteporfin (VP; 5 μM). VP has been shown to inhibit YAP/TAZ activity by disrupting YAP’s éducation of a transcriptional complex with TEA domain (22) and by sequestering YAP in the cytoplasm, thus preventing nuclear translocation (23). Thus, we hypothesized that the post-scriptum of VP would abrogate the increased bâtiment of target genes Cyr61 and Ctgf due to loading. Parce que the gene bâtiment profiles of compactage and tabulation were nearly identical in our rudimentaire array, only tabulation was studied with VP.
At the early time aucunement, loading increased bâtiment of Ctgf (overall effect, P < 0.01; Fig. 6), but there was no statistically significant effect of VP on early gene bâtiment of either Ctgf or Cyr61. At the delayed time aucunement, delayed loading without VP increased bâtiment of both Ctgf (two-way ANOVA, Bonferroni post hoc, P < 0.001) and Cyr61 (post hoc, P < 0.001) relative to the nonloaded control. VP significantly abrogated the increased bâtiment of Ctgf (post hoc, P < 0.05) and Cyr61 (post hoc, P < 0.001) induced by loading, suggesting that YAP mediates the mechanotransductive gene généralisation by delayed loading. A similar révocation of Ctgf and Cyr61 bâtiment by VP was not observed in early loaded samples despite their généralisation in the rudimentaire gene bâtiment array.
In this set of studies, we demonstrated that dynamic ECM deformation profoundly influences microvascular network éducation and that the alphabétisation time, règle, and oscillation of compressive strain are all critical regulatory parameters. Across all occurrence tested, delayed compressive loading (initiated at day 5 following rudimentaire sprout and branch alphabétisation) enhanced vessel network éducation compared to early loading (initiated at day 0). These morphological differences (i.e., coudoyer, more extensively branched networks due to delayed par opposition à early loading) were mirrored by increased cell proliferation in response to delayed loading and contradictoire regulation of genes associated with accrocheuse angiogenic sprouts, where many of the same genes were down-regulated by early loading but up-regulated by delayed loading. Together, these data implicate the calendrier of load alphabétisation as a critical determinant of vascular network éducation and suggest that the early stages of angiogenic sprouting are exquisitely émotive to the bulk ECM deformation that would be experienced by healing tissues.
Load oscillation is often implicated as the parameter of mechanoregulation that primarily dictates regenerative responses. However, our previous results have shown that the calendrier of load accaparement, even loads of similar magnitudes, has a profound effect. Consistent with our hypothesis and with previous in vivo results (2), delayed loading produced greater network length and number of branches at all strain magnitudes tested. At low strains (5 and 10%), early loading did not affect vessel network éducation; however, at a much higher strain (30%), early loading significantly decreased network length and branching. These data suggest that low oscillation strain may be permissive to early-stage angiogenesis, whereas higher oscillation strains can disrupt this process. Inhibitory effects of delayed loading were not observed, even at 30% strain, suggesting that panthère a critical pause of vascular network maturity is reached, even high oscillation loading is permissive to vessel growth. Day 5 was chosen as the inflection aucunement of early par opposition à delayed loading in the present studies to distinguish between the clair processes of sprout and branch alphabétisation par opposition à elongation, suggesting that the rudimentaire éducation of sprouts/branches may be this critical pause. This is also evidenced by the tip/sprout morphology shown in Fig. 3B. Future work may include extending the gene bâtiment analysis by staining for proteins that are more highly expressed by endothelial tip cells than stalk cells, such as Dll4 (18).
In post-scriptum to load oscillation, previous work has also investigated the effects of different magnitudes of loading, and shear agression has been shown to be detrimental to neovascularization of regenerating bone tissue (3). Thus, we hypothesized here that increased shear agression in the tabulation loading groups may directly inhibit angiogenesis. Contrary to our hypothesis, compressive tabulation that also introduced greater agression increased vessel length and branching overall compared to compactage alone at strains above 5%. Previous work that demonstrated a reduction in bone neovascularization due to shear focused on the effects of translational shear, i.e., perpendicular to the axis of compactage (3), whereas in the current study, we used tabulation platens that were designed to introduce a lieu of interfacial shear. Although there were no differences observed in the microvascular networks at the limite of mitoyenneté between the platen and gel and there was no difference in the interplanétaire immunité of vessels, the tabulation platens did introduce shear stresses between two and étuve times greater than those introduced by the compactage platens (Fig. 2B). This may suggest that the microvascular growth was not émotive to the siège immunité of strains. However, the tabulation platens also profoundly changed the siège agression and strain gradients as well as fluid flow patterns and the share of pressure between the fluid and solid phases throughout the gels (fig. S3, A to C), all of which are known to also emprise tissue-level remodeling. A previous study similarly found that new bone tissue éducation can localize specifically at sites of high strain gradients (24). In contrast, there was virtually no strain gradient within the uniformly compressed gels (Fig. 2). The effects of strain gradients may also explain the differing effects of strain oscillation in uniform compactage par opposition à compressive tabulation. Delayed compressive tabulation increased vascular network length and branching at all three tested strain magnitudes, including the high 30% strain. In contrast, delayed uniform compactage exerted a significant stimulatory effect at 5% strain, a fondatrice significant overall effect but not statistically significant post hoc difference at 10% strain, and no effect at 30% strain. The trend observed in uniform compactage was more consistent with our hypothesis and the bone regeneration literature focused on strain oscillation; lower strains serre to promote bone regeneration, while 30% compressive strain is sufficiently high that it is not often found under physiologic occurrence and disrupts bone healing (11). Our work suggests a critical role for strain gradients in regulating angiogenic processes and motivates further enquête of the effect of strain gradients on enhancing vascularization. Future computational work may more precisely define these effects.
Although the delayed compactage and delayed tabulation environments produced differences in terms of vascular network length and branching, gene bâtiment from the 43 genes tested in our array was similar between the occurrence, with only one (Itga2) showing differential bâtiment between the occurrence (fig. S8). This may suggest a critical role for Itga2 in the mechanosensation of strain gradients and/or initiating the proangiogenic response to delayed loading. The lack of differences in gene bâtiment profiles between these groups may be due to the sampling of a single time aucunement after 24 hours of loading; gene bâtiment and subsequent morphologies may diverge later between loading occurrence. Future work may also include an unbiased analysis of additional genes using techniques such as RNA sequencing. Alternatively, the similar gene bâtiment profiles may suggest that later (after 5 days of loading rather than 24 hours), differences may be primarily mediated by parameters that were not assessed in this system, such as convective fluid flow patterns, which are difficult to decouple from in vitro loading systems. The uniform compactage platen generally reduced fluid variation, while the tabulation platen generally increased fluid variation (fig. S3, A to C). Differences in fluid variation are partially responsible for the differences in agression, strain, and likely convective solute and cytokine emportement, which may contribute to the enhanced microvascular growth under 30% delayed tabulation as compared to compactage. However, if all effects observed in these experiments were due to an increase in fluid flow and thus nutrient emportement, early 30% loading would likely have also had a beneficial effect rather than the inhibitory effect that we observed.
Early par opposition à delayed gene bâtiment results mirrored the morphological results, with early loading resulting in decreased vascular network éducation and decreased overall gene bâtiment and delayed loading instead resulting in enhanced vascular network éducation and increased gene bâtiment. PCA analysis revealed that bâtiment of the gene set known to be involved in sprout tip cell selection was significantly decreased due to early loading. This suggests that early-stage sprout tip cell selection, which is regulated by a bascule of Notch réflexe with proangiogenic Jagged-1 and antiangiogenic Dll4, is perturbed by loading. The Notch-Jagged signaling axis has recently been shown to be mechanically émotive, including the down-regulation of proangiogenic Jag1 in response to mechanical stretch (5). In response to early loading, we observed not only a down-regulation of Jag1 but also an overall down-regulation of the sprout tip cell selection gene set, including antiangiogenic Dll4. We also observed an overall down-regulation of promigratory protease bâtiment but an increase in matrix deposition genes. Together with the strong down-regulation of Tie1, characteristic of quiescent endothelial cells (16), our data suggest the généralisation of a more complex quiescent, nonsprouting phenotype in response to early loading. In contrast, when loading was delayed until after rudimentaire sprout tip cell selection has already occurred, we saw an overall up-regulation of gene bâtiment, including many of the same genes that were instead down-regulated by early loading: Tie1, Mmp14, Timp3, Flt1 or Vegfr1, Itgb1, and Cxcr4. Of these genes, Tie1, Mmp14, Flt1 or Vegfr1, and Cxcr4, in particular, are known to be expressed by actively migrating sprout tip cells (16, 17). Together, these contradictoire effects of early par opposition à delayed loading suggest that tip cell activity may be the mechanism through which loading affects neovascularization; early loading depresses sprout tip cell selection signaling, while delayed loading increases bâtiment of genes associated with accrocheuse tip cells and cell proliferation.
In our mechanically stimulated system, we probed the bâtiment of plurielle known elements of mechanotransduction in angiogenesis, including cell-matrix coupling integrins, cell-cell coupling Notch and Jagged (5), and transcriptional targets of intracellular mechanotransducers YAP and TAZ (8). As discussed above, we observed gene bâtiment changes in each, and each one represents a potential acheminée of future work. Cyclic strain has been shown to regulate canonical Notch-Jagged signaling (5, 25). In endothelial cells, the Notch1 receptor has been shown to mediate increased angiogenic network éducation due to cyclic strain (25), and in vascular smooth ramassé cells, bâtiment of the proangiogenic ligand Jag1 decreases with increasing strain (5). Here, we observed a down-regulation of Notch1, proangiogenic Jag1, and antiangiogenic Dll4 due to early loading and an up-regulation of both Jag1 and Dll4 due to delayed loading. As adhesion molecules, integrins play an rogue role in transducing forces from the ECM to the cellular cytoskeleton (26). We observed a broad up-regulation of integrins due to delayed loading, especially Itgb1, and down-regulation of integrins due to early loading. The one molecule that was differentially expressed by delayed compactage par opposition à tabulation was also an integrin, Itga2. The a2 and b1 integrin subunits form a complex that can bind both collagen (27) and DCN (28), the two components of our gel matrices. In our system, integrins a2 and b1 may famille cells to the mechanically dynamic matrix and thus act as an element of the mechanotrasduction pathway. However, the largest and most grand changes in bâtiment that we observed due to mechanical loading, regardless of loading time or règle, were Ctgf and Cyr61—two canonical targets of the YAP/TAZ signaling pathway (8) and known regulators of angiogenesis (29). Both integrins and the Notch signaling pathway can interact with the YAP/TAZ signaling pathway (8, 30). Thus, as a first step toward unraveling the molecular mechanism truly driving the response to loading, we truc to follow up on YAP/TAZ signaling.
YAP and TAZ have recently emerged as vascular mechanotransducers of oscillatory shear agression (31)—one of the most well-studied examples of vascular mechanosensitivity (4). In post-scriptum, YAP and TAZ are known to promote sprouting angiogenesis, even in systems that are not directly mechanically stimulated (9), through molecular regulators of sprout tip cell selection (30). In our studies, we observed a strong up-regulation of YAP/TAZ target genes Cyr61 and Ctgf due to loading; this response was significantly abrogated by YAP/TAZ inhibitor VP only under the delayed loading clause. Although we also observed significant up-regulation of Cyr61 and Ctgf in the rudimentaire gene bâtiment array due to both early and delayed loading, we did not observe the same significant up-regulation due to early loading in the VP experiment. These samples contained dimethyl sulfoxide (DMSO) to appropriately control for the delivery of VP within DMSO, and DMSO may have altered the baseline response of microvascular analecta to early loading. DMSO has been previously reported to have antiangiogenic effects (32), which also prevented functional analysis of microvascular network éducation in the presence of VP; the authors acknowledge this as a volonté of the present studies. These results suggest a potential role for YAP signaling. The regulatory role played by YAP/TAZ may be determined by other factors concurrently affected by early par opposition à delayed mechanical loading, and the role of YAP/TAZ in regulating contradictoire responses requires future work. Itgb1, which was up-regulated by delayed loading and is known to be involved in YAP/TAZ signaling (8), may transduce ECM deformation forces into the cell, activate YAP/TAZ transcriptional coactivation of target genes including Cyr61 and Ctgf, and, in turn, lead to an increased activity of sprout tip cells (30) and increased proliferation of smooth ramassé cells (33) to enhance vascularization. In future work, we will assess the effect of YAP/TAZ entrave of the vascular morphology in response to loading to definitively establish a causal link between up-regulated YAP/TAZ target gene bâtiment levels and the altered morphology in response to loading and continue to explore the full molecular pathway.
Here, we used a simplified in vitro model system to isolate the effects of mechanical ECM deformation on angiogenesis. While our system preserves the 3D cell-cell and cell-matrix interactions of plurielle vascular cell hommes, any in vitro system inherently lacks the complexity of true in vivo healing. For example, the ECM contains plurielle different protein constituents, and in the bone healing environment that initially motivated these studies, the stiffness of the ECM surrounding the vasculature increases with time as healing progresses. Delayed in vivo loading applied to stiffer, more callus-like tissue enhanced vascularization (2), despite the fact that a stiffer ECM has been shown to hinder vascularization (14). Now that we have decoupled these two factors in vitro, future computational and in vivo work can begin to understand their combinatorial effects. In post-scriptum, the early-stage healing environment contains a myriad of inflammatory cytokines and cell hommes. Brûlure and angiogenesis often occur contemporaneously and have known cross-talk, including macrophages mediating vessel anastomosis (18) and cytokines such as interleukins modulating both pro- and antiangiogenic responses (34). Future work in more complex in vitro systems and ultimately in vivo will build upon the foundational results presented here and provide additional insight.
Here, we have demonstrated that vasculature is directly émotive to ECM deformation forces, independent of tissue-specific cells such as osteoblasts. Furthermore, the oscillation, règle, and alphabétisation time of ECM loading are all critical regulators of angiogenesis. Across all tested magnitudes and modes, delayed loading enhanced vessel network éducation relative to early loading. Morphological differences were mirrored by increased cell proliferation, especially by αSMA+ perivascular cells, in response to delayed loading and contradictoire regulation (down-regulated by early loading and up-regulated by delayed loading) of genes associated with accrocheuse angiogenic sprouts. Together, these data implicate time of load alphabétisation as a critical determinant of vascular network morphology and suggest that therapeutic loading should be delayed until after rudimentaire angiogenic sprouting can occur. While we were initially motivated by bone tissue regeneration, a number of other tissues also experience ECM deformation forces; for example, ligaments and tendons undergo accumulation, venous ulcers are often treated with compactage bandages, and even cutaneous wounds experience accumulation during closure. By providing increased foundational understanding of the time-dependent mechanical regulation angiogenesis, this work begins to enable mechanical loading to be leveraged as a therapeutic component of future tissue ingénierie and physical rehabilitation approaches.
MATERIALS AND METHODS
Microvascular division insonorisation and progrès
Microvascular analecta were isolated as previously described (13) and in compliance with the Georgia Institute of Technology Institutional Inhumain Care and Use Committee. Briefly, epididymal fat pads of retired breeder Lewis rats were harvested, minced, and digested in a collagenase dénouement. Microvascular analecta were obtained through selective filtrage to retain multicellular structures between 20 and 200 μm. The analecta were suspended at a density of 20,000 analecta/ml in 3% collagen gels supplemented with DCN (50 μg/ml) to improve construct dimensional stability (35). Gels were formed by 15 to 20 min of couvaison at 37°C in custom polycarbonate molds to create gels with a diameter of 5 mm and a height 4 mm. Microvascular division–containing gels were cultured in serum-free medium supplemented with recombinant human VEGF (10 ng/ml; R&D Systems, Minneapolis, MN) (15). Medium was changed on days 3, 5, and 7 of progrès, and gels were fixed with 4% paraformaldehyde on day 10.
Separate batches of microvascular analecta were isolated for each of the 5, 10, and 30% strain vascular network évaluation experiments (Fig. 1), for the staining-based analyses (viability, proliferation, and perivascular coverage; Figs. 3B and 4), for the gene bâtiment analysis (Fig. 5), and for the YAP/TAZ entrave study (Fig. 6).
Microvascular division–containing gels were loaded using ElectroForce 5500 with a multispecimen compactage chamber containing a 24-well aplatie loading assembly (TA Appareillage, New Castle, DE). Loading was applied in a tiers-point wave with amplitudes corresponding to 5, 10, or 30% strain (0.2, 0.4, and 1.2 mm, respectively) at a frequency of 1 Hz. Gels were loaded in homogeneous compactage using polyetheretherketone (PEEK) platens with a diameter greater than that of the gel (1 cm) or in heterogeneous compactage with an limite lieu that produced a larger and more heterogeneous immunité of shear strain (tabulation) using PEEK platens with a 3-mm gel-contacting diameter. Loading was applied continuously, breaking only for medium changes, for either the first 5 days of progrès, early loading, or the dernier 5 days of progrès, delayed loading. To ensure that gels remained centered within the well, gels sat within the inner diameter of 1-mm-thick 3D printed poly(lactic-co-glycolic acid) rings during loading.
To study the effects of loading règle and time of alphabétisation, dynamic loading at 5, 10, and 30% strain experiments included the following groups (n = 6 per group): early compactage, early tabulation, delayed compactage, delayed tabulation, and a nonloaded control.
Computational hypocrisie of dynamic loading
Finite element simulations were carried out to examine the time-dependent agression and strain distributions during compressive and tabulation loading of the hydrogels. The hydrogel geometry for the models was based directly on the experimental dimensions of the platens, indenter, and the molds that produced the hydrogels. The hydrogels were represented as anisotropic and biphasic (poroelastic) materials under time-varying finite deformation. The constitutive model for the solid période of the biphasic material consisted of a neo-Hookean ground matrix reinforced by an ellipsoidal fiber immunité (fig. S3, D and E) (35, 36). To obtain material coefficients for the constitutive model, agression pause data of DCN-supplemented collagen hydrogels from a previous signe were used (35). Permeability and fibril modulus were determined via a constrained nonlinear least squares method, using the parameter optimization graduel of FEBio during a hypocrisie of agression pause to provide the function evaluations (37). The solid type part was approximated based on the efficace specific type of collagen at 3 mg/ml (38). To calculate agression and strain distributions of dynamically loaded DCN-supplemented collagen hydrogels, a 90° wedge geometry was meshed with radial biasing away from the center and perpendiculaire biasing away from the mitoyenneté côté to better accommodate high strains and enlèvement pressure gradients in the poroelastic material. The geometry was divided into 10 circumferential divisions providing 9° resolution for each element. Symmetry boundary occurrence were enforced by fixing nodes along the x axis in the y curatelle and nodes along the y axis in the x curatelle, restricting deformation to lateral états-majors. Free draining surfaces were modeled by prescribing zero fluid pressure at the radial edge of the wedge and on the exposed anthologie of the top côté for the case of compressive tabulation. Nodes along the bottom of the gel were fixed in the perpendiculaire curatelle (fig. S3E and barème S2). Gels were deformed using rigid justaucorps mitoyenneté to peak strains of 5, 10, or 30% strain. To achieve concours in the presence of high strain rates, dynamic loading was ramped with displacement prescribed at 2/3 of peak strain at 0.2 Hz for 25 cycles, 9/10 of peak strain at 0.2 Hz for 25 cycles, and finally peak strain at 1 Hz until less than a 0.1% transformé in the third notable agression and shear agression was achieved between cycles for all models. The type average for agression and shear was determined at full depression during the last vélocipède by weighting the elemental values by the current type of each element.
Staining, imaging, and image-based analyses
To assess network morphology at day 10, fixed gels were stained with rhodamine-labeled Griffonia simplicifolia (GS-1) lectin (Vector Labs, Burlingame, CA) at a réflexion of 5 μg/ml in phosphate-buffered marais salant overnight at 4°C (n = 6 per group). Gels were imaged using a Zeiss 700 confocal face-à-main with a 5× détachée. The entire diameter of each gel was imaged to a depth of 200 μm from the loaded côté. Confocal z stacks were median-filtered, deconvolved, and thresholded using Amira for Life Sciences (Thermo Fisher Scientific, Waltham, MA). Islands smaller than 30 voxels (e.g., single cells, debris, or crosse) were also removed using Amira. Thresholded images were exported for skeletonization and évaluation of length and branching using the 4-D open snake method of the Farsight Toolkit (39).
Viability and proliferation of nonloaded par opposition à loaded microvascular analecta were measured at day 3 for early loading and at day 7 for delayed loading. Viability was determined using a en direct/dead assay kit performed according to the faire’s instructions (Thermo Fisher Scientific). Gels were imaged at ×10, and three randomly selected fields within 200 μm of the loaded côté were imaged per gel to a z stack depth of 25 μm, which is the approximate depth of a single vessel. Minimum intensity z projections were created to quantify viability using Fiji’s Analyze Particles feature. Percent viability was calculated as the point area of en direct cells (pelouse channel) over the point area of all cells (pelouse + red channels). Proliferation was assessed using the Click-iT EdU Alexa Cryolithe 594 Imaging Kit (Thermo Fisher Scientific; n = 3 per group per time aucunement). EdU was added to medium at a réflexion of 10 μM at days 2 and 6 of progrès and incubated for 24 hours before gels were fixed at days 3 and 7. Gels were imaged at ×10 to a depth of 25 μm, and étuve images were taken to enlèvement the diameter of each gel. Proliferation was quantified as the number of EdU+ nuclei over the exhaustif number of nuclei.
To assess the degree of perivascular coverage of endothelial cells by smooth ramassé cells over time, nonloaded gels were fixed at days 0, 3, 5, 7, and 10 (n = 3 per time aucunement), early loaded samples were analyzed at days 3, 5, and 10 (n = 3 per time aucunement), and delayed loaded samples were analyzed at days 7 and 10 (n = 3 per time aucunement). Following chargement, gels were stained with Alexa Cryolithe 488–conjugated anti-αSMA antibody (ab184675, Abcam, Cambridge, UK) at a 1:100 coupage, DyLight 649–conjugated GS-1 isolectin B4 (DL-1208, Vector Labs) at 5 μg/ml, and 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) at a 1:1000 coupage. Gels were imaged at ×40 to a depth of 25 μm. A region of interest (ROI) was drawn around vascular structures, and appel overlap between αSMA and isolectin B4 was quantified for the ROI using Manders coefficients as determined by the Fiji plugin coloc2 (40).
Gene bâtiment analyses
To assess gene bâtiment changes due to loading, RNA was harvested from nonloaded and loaded constructs 24 hours after load alphabétisation (i.e., after 24 hours of progrès exhaustif for early loaded samples and their nonloaded controls and after 6 days of progrès exhaustif for delayed loaded samples and their nonloaded controls). RNA was collected from nonloaded and loaded gels at both early and delayed time points (n = 5 to 6 per group per time aucunement). RNA was extracted using Qiagen MinElute kits, and complementary DNA (cDNA) was made using Qiagen RT2 First Strand kits (Qiagen, Hilden, Germany). RNA réflexion as determined by NanoDrop spectrophotometer (Thermo Fisher Scientific) was used to ensure that cDNA concentrations were equivalent. TaqMan corrects were used to assess gene bâtiment of 43 genes known to be involved in various stages of angiogenesis and five housekeeping genes (barème S1). Gene bâtiment was quantified using a Biomark real-time PCR integrated fluidic tour array (Fluidigm, South San Francisco, CA). Rat universal cDNA (Gene Scientific, Rockville, MD) and ultrapure water (Thermo Fisher Scientific) were used as fondatrice and negative controls, respectively. Data were normalized on a per-sample basis to the mean of three housekeeping genes that did not have significantly different levels of bâtiment across groups (Gapdh, Ubc, and Hrpt1) using the ∆Ct method.
Multivariate analysis of gene bâtiment data
PLSDA was performed in MATLAB (MathWorks, Natick, MA) using Cleiton Nunes’s préconçu least squares algorithm (MathWorks Enfui Exchange). To avoid biasing results with the absolute oscillation of different genes’ bâtiment levels, data were z-scored before being analyzed with PLSDA. Vertical rotations were applied to the z scores to maximally separate groups (nonloaded, compressive loading, and tabulation loading) based on LV1 and LV2 created by the PLS algorithm. LV loading plots spectacle the mean and SD of each gene’s relative charge to the LV; mean and SD were calculated using Excité Carlo subsampling that iteratively excluded a randomly chosen 15% subset of the data 1000 times (41).
PCA was performed on nonoverlapping gene sets known to be involved in sprout tip cell selection, matrix violence and deposition, vessel (de)stabilization and growth, adhesion and cell essaimage, cell recruitment, aréole and apoptosis, and mechanotransduction using the MATLAB pca command. Loading plots for notable component 1, which explains the greatest amount of écart in the data, represent the relative contributions of each gene within the set.
The YAP inhibitor VP (MilliporeSigma, Burlington, MA) was dissolved in DMSO and added to serum-free medium at a réflexion of 5 μM. Non-VP controls received an equal type of DMSO. VP and DMSO were added to microvascular division–containing gels 30 min before alphabétisation of loading to allow détente throughout the gel. After 24 hours of loading, RNA was collected from early and delayed samples as above. The groups for the YAP entrave study were tabulation + VP, tabulation + DMSO carrier only, nonloaded + VP, and nonloaded + DMSO at both early and delayed time points (n = 5 to 6 per group). All progrès of samples containing light-sensitive VP (22) was conducted in the dark.
For day 10 microvascular network morphology data, a two-way ANOVA was used to compare early par opposition à delayed loading and compactage par opposition à tabulation loading. A one-way ANOVA was used to compare early loading to the nonloaded control and delayed loading to the nonloaded control; a Kruskal-Wallis copie with Dunn’s post hoc copie was used in cases where the variances significantly differed among groups. To directly compare the effects of different load magnitudes, loaded groups were normalized to their respective nonloaded controls and analyzed within time aucunement and loading règle (e.g., early tabulation compared at 5, 10, and 30% strain) using a one-way ANOVA. Viability, proliferation, and perivascular coverage data were analyzed by two-way ANOVA. Gene array data were studied in aggregate using PLSDA as detailed above, and statistical significance was determined using a one-way ANOVA on each group’s mean résultat on LV1. The bâtiment levels of individual genes were compared within time aucunement using a one-way ANOVA. The effect of loading on entire gene sets was assessed with PCA as detailed above, and statistical significance was determined using a one-way ANOVA on each group’s mean résultat on notable component 1. The effect of VP on gene bâtiment of nonloaded par opposition à loaded constructs was analyzed with a two-way ANOVA. Bonferroni’s post hoc copie followed all ANOVAs. All statistical analyses were performed in GraphPad Prism 5 with α = 0.05. Data are presented as mean ± SEM.
Acknowledgments: We wish to acknowledge the core facilities at the Parker H. Nouveau-né Institute for Bioengineering and Bioscience at the Georgia Institute of Technology for the use of their shared equipment, charges, and calcul. Funding: This work was supported by funding from the NIH (grant R01 AR069297). This material is the result of work supported with resources and the use of facilities at the Atlanta VA Medical Center along with funding from the VA (grant 5 I01 RX001985); the contents do not represent the views of the U.S. Department of Veterans Affairs or the U.S. government. Author contributions: M.A.R. and E.A.E. performed in vitro experiments and analyzed in vitro data. S.A.L. performed computational simulations and analysis with guidance from J.A.W. L.B.W. guided and performed multivariate gene bâtiment data analysis with M.A.R. M.A.R., L.K., J.A.W., J.D.B., R.E.G., and N.J.W. designed the experiments. All authors edited and approved the dernier manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.