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NFATc1 transcription factor is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis and osteoclastogenesis. We identified a role for calcineurin-NFAT signaling in lymphatic development and patterning. NFATc1 was colocalized with lymphatic markers Prox-1, VEGFR-3 and podoplanin on cardinal vein as lymphatic endothelial cells (LEC) are specified and as they segregate into lymph sacs and mature lymphatics. In NFATc1 null mice, Prox-1, VEGFR-3 and podoplanin positive endothelial cells sprouted from the cardinal vein at E11.5, but poorly coalesced into lymph sacs. NFAT activation requires the phosphatase calcineurin. Embryos treated in utero with the calcineurin inhibitor cyclosporine-A showed cytoplasmic NFATc1, diminished podoplanin and FGFR-3 expression by the lymphatics and irregular patterning of the LEC sprouts coming off the jugular lymph sac, which suggests a role for calcineurin-NFAT signaling in lymphatic patterning. In a murine model of injury-induced lymphangiogenesis, NFATc1 was expressed on the neolymphatics induced by lung-specific overexpression of VEGF-A. Mice lacking the calcineurin Aβ regulatory subunit, with diminished nuclear NFAT, failed to respond to VEGF-A with increased lymphangiogenesis. In vitro, endogenous and VEGF-A-induced VEGFR-3 and podoplanin expression by human microvascular endothelial cells was reduced by siRNA to NFATc1, to levels comparable to reductions seen with siRNA to Prox-1. In reporter assays, NFATc1 activated lymphatic specific gene promoters. These results demonstrate the role of calcineurin-NFAT pathway in lymphangiogenesis and suggest that NFATc1 is the principle NFAT involved.
The development of specialized endothelial cells (arterial, venous and lymphatic endothelium) requires cell lineage selection through the expression of specific subsets of genes. Evidence from mouse and zebra fish models have identified signaling pathways such as Notch and COUP-TFII that direct endothelial cell fate toward the arterial and venous endothelial lineages (Niessen and Karsan, 2007; You et al., 2005). However, little is known about the differentiation transition points and regulatory mechanisms of lymphatic endothelial lineage selection and maintenance.
The lymphatic vasculature regulates interstitial fluid balance and facilitates immune responses by transporting lymphocytes and macrophages (Oliver, 2004). Lymphatic malformations represent dysfunctional development of this vascular compartment and are the most common form of congenital vascular defects (Lee et al., 2005). In mouse, lineage selection of differentiation-competent lymphatic endothelial cells (LEC) occurs within the cardinal vein from embryonic day (E) E9.0–E10.0, a process dependent on the transcription factors and Prox-1 (Wigle and Oliver, 1999) and Sox-18 (Francois et al., 2008). As the lymphatic endothelium develops, LEC are distinguished from blood endothelial cells (BEC) by expression of genes including Prox-1 (Wigle and Oliver, 1999) podoplanin (Schacht et al., 2003), neuropilin-2 (Yuan et al., 2002), angiopoietin-2 (Gale et al., 2002), VEGFR-3 (He et al., 2002), SYK and SLP76 (Sebzda et al., 2006).
NFAT transcription factors play a critical role in lineage specification during T lymphocyte Th1/Th2 differentiation (Peng et al., 2001; Porter and Clipstone, 2002), osteoclast differentiation (Ishida et al., 2002; Matsuo et al., 2004) and endothelial cell differentiation in cardiac valve morphogenesis (Chang et al., 2004; de la Pompa et al., 1998; Lee et al., 2006; Ranger et al., 1998). The NFAT family includes five proteins designated as NFATc1-NFATc5 and often exhibit functional redundancy (Hogan et al., 2003). In the non-active state, NFATs are phosphorylated and remain cytosolic. NFAT activation requires dephosphorylation by the calcium dependent serine/threonine phosphatase calcineurin, a heterodimer of a catalytic subunit A and a regulatory subunit B. Dephosphorylated NFATs translocate into the nucleus to regulate transcription (Hogan et al., 2003).
Cell activation by a variety of factors including vascular endothelial growth factor-A (VEGF-A) boosts intracellular calcium, activates calcineurin and induces NFAT transcriptional activity (Armesilla et al., 1999; Schweighofer et al., 2007). Transcriptional targets of NFATs in the endothelium include tissue factor, Down syndrome critical region 1 (DSCR-1), E-selectin, Cox-2 and VCAM-1 (Hesser et al., 2004; Lange et al., 2004; Minami et al., 2006). Mice null for NFATc3 and NFATc4 as well as mice lacking the calcineurin phosphatase activity show defective angiogenesis and vascular patterning (Graef et al., 2001). However, function of the NFAT/calcineurin pathway in lymphatic vascular development has not been studied. Using in vitro and in vivo systems, we demonstrate through loss of function and gain of function analyses that calcineurin-NFAT signaling, specifically through NFATc1, is a determinant of lymphatic endothelial cell patterning in developmental and injury-induced lymphangiogenesis.
To understand the role of NFATc1 in lymphatic development, detailed analyses of NFATc1 expression on the murine lymphatic vasculature was performed beginning at E10.5 when the lymphatics first sprout from the cardinal vein. LEC were defined by expression of Prox-1, VEGFR-3 and podoplanin (Wigle and Oliver, 1999). The pan-endothelial isolectin GSL-B4 (Akeson et al., 2003; Ponder et al., 1985) was used to identify all endothelial cells. At E10.5 regions of cardinal vein where lymphatic sprouting was active were identified by unilateral Prox-1 expression (Fig. 1A). These endothelial cells also expressed NFATc1 (Fig. 1A and inset). Some cardinal vein endothelial cells were positive for NFATc1 alone. Prox-1 alone was detected in developing liver cells (Fig. 1A). However, NFATc1 and Prox-1 were colocalized only in the regions of active lymphatic sprouting.
At E12.5, when the lymphatic sprouts segregate from cardinal vein to form lymph sacs, NFATc1 (Fig. 1B, Supplemental Fig. 1A) and Prox-1 (Fig. 1C, Supplemental Fig. 1B) were uniformly expressed in developing lymph sacs, unlike the unilateral expression on cardinal vein at E10.5 (Fig. 1A). NFATc1 (Fig. 1B, Supplemental Fig. 1A) colocalized with Prox-1 (Fig. 1B, Supplemental Fig. 1B) as well as the lymphatic markers VEGFR-3 and podoplanin on the developing jugular lymph sacs and the LEC sprouting from the lymph sacs (Fig. 1D, 1E, Supplemental Fig. 1C–1D). At this time, the cardinal vein endothelial cells expressed NFATc1 and Prox-1, but were not positive for LEC markers VEGFR-3 (Fig. 1D) or podoplanin (Fig 1E). The jugular vein endothelium was negative for Prox-1, VEGFR-3 and podoplanin (Fig. 1C–1F). However, a few endothelial cells on the jugular vein at E12.5 were NFATc1 positive (Fig. 1B, 1D, 1F). GSL-B4 staining confirmed that only the endothelial cells of cardinal vein, developing lymph sacs and lymphatic sprouts from the sacs, but not BEC, coexpressed NFATc1 and Prox-1 (Fig. 1F, Supplemental Fig. 1E).
The lymphatic vasculature is distributed throughout the mouse embryo at E14.5 and LEC are undergoing terminal differentiation to form mature lymphatics (Oliver, 2004). Analysis of trunk, pulmonary and dermal lymphatics at E14.5 revealed that NFATc1 was coexpressed with Prox-1, VEGFR-3 (Fig. 1G–I) and podoplanin (Supplemental Fig. 1F, 1H) on LEC. NFATc1 was coexpressed with Prox-1 and VEGFR-3 on lymphatics of adult tissues as well (Fig. 1J). During embryogenesis, NFATc3 is expressed on the perivascular tissues that guide the endothelial cells in developmental angiogenesis (Graef et al., 2001), but was not detected on developing lymphatic system (Supplemental Fig. 1J, 1K). These results demonstrate that NFATc1 is expressed on LEC along with the essential lymphatic transcription factor Prox-1 throughout lymphatic development. Further, NFATc1 was localized to nuclei at each stage of lymphatic development suggesting it is in its active, dephosphorylated form (Ranger et al., 1998). These data suggest that NFATc1 participates in lymphatic development, patterning and maintenance of lymphatic vasculature.
NFATc1 null mice typically die due to heart valve defects (Ranger et al., 1998) at about E12.5. However, several studies (de la Pompa et al., 1998; Phoon et al., 2004; Ranger et al., 1998) suggest that the embryonic blood vascular endothelium and the embryonic-placental vascular systems are normal in NFATc1 null embryos. Therefore, to gain insight into the role of NFATc1 in early embryonic lymphatic development, NFATc1 null mice (Ranger et al., 1998) were analyzed at E11.5. Cardinal vein endothelial cells and the lymphatic sprouts in wild type as well as NFATc1 null mice coexpressed Prox-1, VEGFR-3 and podoplanin (Fig. 2A, 2C, Supplemental Fig. 2A, 2B). Staining with GSL-B4 showed typical blood vasculature morphology in wild type (Fig. 2B) and NFATc1 null mice (Fig. 2D) at E11.5. LEC, identified by Prox-1 staining, migrated and coalesced into lymph sacs as they sprouted from the cardinal vein in wild type (Fig. 2A, 2B, 2E) and NFATc1 heterozygote mice (Fig. 2F). While endothelial cells sprouted from the cardinal vein in NFATc1 null mice, fewer Prox-1 positive cells were organize into vessel-like sacs and there appeared to be more Prox-1 cells dispersed as single cells or in small clusters within the mesenchyme adjacent to the cardinal vein (Fig. 2C, 2D, 2G). Morphometric analysis determined there was no difference in the average area of the cardinal vein (Fig. 2H), the number of developing lymph sacs per μm2 (Fig. 2I) or the number of Prox-1 positive sprouting LEC (Fig. 2J) in wild type, NFATc1 heterozygote and NFATc1 null mice. However, the luminal area of developing lymph sacs was reduced by 60% (±4%) (Fig. 2K) and the number of Prox+cells/lumen was reduced by 40% (±2.9%), (Fig. 2L) in NFATc1 null compared to wild type animals. For all parameters measured there was no difference between wild type and NFATc1 heterozygotes (Fig. 2H–2L). These data suggest that NFATc1 regulates lymphatic endothelial cell patterning and lymph sac formation, but not sprouting from cardinal vein in early development.
As NFATc1 null mice die early in gestation before the lymphatic vasculature has fully developed, we used the calcineurin inhibitor cyclosporine-A (CsA) to block NFAT activity in utero. CsA blocks the function of the phosphatase calcineurin, diminishing dephosphorylation, activation and nuclear translocation of NFAT (Chang et al., 2004; Graef et al., 2001). CsA crosses the placental barrier and inhibits calcineurin activity in the fetal mice (Graef et al., 2001). Calcineurin activity is required for blood vascular development through E8.5 (Graef et al., 2001) and for endocardial valve elongation in heart through E12.0 (Chang et al., 2004). Therefore, embryos treated with CsA from E12.5 to E14.5, after blood vascular development and cardiac valve elongation is complete, were used to study the effects of calcineurin-NFAT inhibition on subsequent lymphatic patterning and expansion.
At E14.5, the jugular lymph sacs are separated from the Prox-1 and VEGFR-3 negative blood vasculature (Fritz-Six et al., 2008). Analysis of vehicle-treated control embryos showed that endothelial cells of the jugular lymph sac expressed Prox-1, VEGFR-3 (Fig. 3A), nuclear NFATc1 (Fig. 3A, 3B), podoplanin (Fig. 3C, 3D, Supplemental Fig. 3A) and FGFR-3 (Fig. 3M), a Prox-1 regulated lymphatic gene (Shin et al., 2006). With CsA treatment the size and morphology of the jugular lymph sacs was unaffected (Fig. 3A–3H, Supplemental Fig. 3A–3D) and the endothelial cells expressed Prox-1 and VEGFR-3 (Fig. 3E). However, NFATc1 was cytoplasmic (Fig. 3E, 3F) and expression of podoplanin (Fig. 3G, 3H, Supplemental Fig. 3B–3D) and FGFR-3 (Fig. 3N, 3O) were diminished. Compared to vehicle treated embryos (Fig. 3I, 3J), Prox-1 and VEGFR-3 double positive LEC sprouts from the jugular lymph sac were poorly organized (Fig. 3K) and showed diminished podoplanin (Fig. 3L) expression in CsA treated embryos. The trunk (Supplemental Fig. 3E–3J) and dermal lymphatics (Supplemental Fig. 3K–3N) also had diminished podoplanin expression. GSL-B4 staining confirmed that the blood vasculature was unaffected by CsA treatment (Fig. 3, Supplemental Fig. 3). The CsA treated embryos also showed generalized fluid accumulation or edema, implying a diminished lymphatic function (Supplemental Fig. 3O–3Q). These in vivo results support a role for calcineurin-NFAT signaling in lymphatic development and patterning.
We have previously reported that VEGF-A overexpression in the lungs of SP-CrtTA/tetOVEGF-164 bitransgenic mice promotes distal lung lymphangiogenesis (Mallory et al., 2006). We found strong NFATc1 expression within the VEGF-A-induced pulmonary lymphatic vasculature (Fig. 4). To examine the role of calcineurin-NFAT pathway in VEGF-A-induced pulmonary lymphangiogenesis, we bred the SP-CrtTA/tetOVEGF-164 mice with CnAβ null mice, lacking the β regulatory subunit of calcineurin A. CnAβ null mice have diminished NFAT activation and nuclear localization. While, these mice are viable, they fail to respond to increased stress such as cardiac ischemia and acute inflammation (Bueno et al., 2002a; Bueno et al., 2002b).
We hypothesized that CnAβ may play a role in VEGF-A-induced lung lymphangiogenesis, a condition mimicking lung injury and stress. We predicted that dysregulation of calcineurin would diminish NFATc1 activation and reduce VEGF-A-induced VEGFR-3 expression in distal lung. Dams bearing wild type, CnAβ null, SP-CrtTA/tetOVEGF-164 and CnAβnull/SP-CrtTA/tetOVEGF-164 were treated with doxycycline from E14.5 to E18.5 to induce VEGF-A expression. Using immmunohistochemistry and morphometric analysis of lung area (Bolender et al., 1993) we determined there was no change in Prox-1 or VEGFR-3 expression between wild type mice, single transgenic SP-CrtTA or tetOVEGF-164 and CnAβ null mice (data not shown). However, VEGF-A induction increased NFATc1 by 1.8-fold (±0.22) (Fig 4A–4C), Prox-1 by 4.0-fold (±0.74) and VEGFR-3 by 2.2-fold (±0.03) in the distal lungs of SP-CrtTA/tetOVEGF-164 compared to lungs from CnAβ null mice (Fig. 4D, 4E). For CnAβnull/SP-CrtTA/tetOVEGF-164, with inhibition of calcineurin and VEGF-A induction, Prox-1 expression was not affected (Fig. 4D), but NFATc1 was reduced 3.5-fold (±0.07) (p=0.0041, Fig. 4C) and VEGFR-3 was reduced 1.6-fold (±0.01) (p=0.023, Fig. 4E) compared to SP-CrtTA/tetOVEGF-164 mice. These in vivo results show that, activation of the calcineurin/NFAT pathway contributes to lymphangiogenesis in a model of VEGF-A induced pulmonary injury.
Activation of VEGFR-2 by VEGF-A increases intracellular calcium leading to calcineurin activation and nuclear translocation of NFATc1 in human pulmonary valve endothelial cells in culture (Johnson et al., 2003; Lee et al., 2006). We assessed NFATc1 nuclear translocation in response to VEGF ligands in primary HMVEC-L cells, derived from human adult lungs. HMVEC-L are a mixed population of BEC and LEC. Characterization of HMVEC-L by flow-cytometry showed that 98% of cells expressed both VEGFR-3 and podoplanin (Fig. 5A) indicating a predominant lymphatic population. For the HMVEC-L shown in Figure 5A, 35% of HMVEC-L were Prox-1 positive and 62% Prox-1 and NFATc1 positive, (Fig. 5B and 5C). To determine whether VEGF-A promoted nuclear translocation of NFATc1, HMVEC-L were first factor starved in EGM-1% overnight leading to a decrease in the number of cells with nuclear NFATc1 from 62% to 18% of cells. Treatment of HMVEC-L with VEGF-A-165 (20 ng/ml) induced strong nuclear localization of NFATc1with peak induction at 2 to 3 hours in up to 71% of cells (Fig. 5D). Pre-treating HMVEC-L with 0.2 μM CsA for 30 minutes before VEGF-A-165 treatment reduced NFATc1 nuclear translocation to 18% of cells (Fig. 5D).
Ligands VEGF-C and VEGF-D support proliferation, migration and survival of LEC in culture through activation of VEGFR-3 (Makinen et al., 2001). To determine whether activation of VEGFR-3 induces NFATc1 nuclear translocation, factor-starved HMVEC- L were cultured for 3 hours with VEGF-C, VEGF-D or VEGF-C C156S, a mutant of VEGF-C that activates VEGFR-3, but not VEGFR-2 (Joukov et al., 1998), at concentrations up to 500ng/ml. While VEGFR-3 was phosphorylated (data not shown), VEGFC, VEGF-D and VEGFC156S did not induce NFATc1 nuclear localization (Fig. 5E). Activation of VEGFR-3 does not directly induce cytoplasmic calcium influx (Olsson et al., 2006). As calcium is required for calcineurin activity, this may explain why activation of VEGFR-3 failed to induce nuclear NFATc1. The mature forms of VEGF-C and VEGF-D can activate VEGFR-2, although with binding affinities three-fold lower than for VEGFR-3 (Achen et al., 1998; Joukov et al., 1997). The short in vitro culture conditions in our system may be insufficient for proteolytic processing of VEGF-C and VEGF-D and may explain the failure to activate VEGFR-2 and NFATc1 translocation. These data suggest that in this model of VEGF-A-induced lymphangiogenesis, NFATc1 nuclear localization is initiated through VEGFR-2 and not VEGFR-3.
To evaluate the role of NFATc1 in the maintenance of lymphatic endothelial lineage, we used siRNA to examine whether the loss of NFATc1 alone or in combination with Prox-1, affected the expression of LEC genes VEGFR-3 and podoplanin. When transfected into HMVEC-L, siRNA against NFATc1 decreased NFATc1 protein levels without affecting the levels of Prox-1 and siRNA against Prox-1 decreased the endogenous levels of Prox-1 without change in the NFATc1 levels (Fig. 6A), indicating that there was no reciprocal regulation between Prox-1 and NFATc1. NFATc1 siRNA did not affect the expression of other NFAT family members, thus establishing target specificity (Fig 6 B). Both NFATc1 and Prox-1 siRNAs reduced VEGFR-3 and podoplanin, without affecting VEGFR-2 (Fig. 6C). Quantification of western blots showed that NFATc1 siRNA significantly decreased VEGFR-3 levels by 69% (p=0.0001) (Fig. 6C, 6D) and podoplanin levels by 71% (p=0.0001) (Fig. 6C, 6E). Comparable levels of inhibition were achieved with siRNA for Prox-1, which reduced VEGFR-3 levels by 78% (p=0.020) (Fig. 6C, 6D) and podoplanin levels by 62% (p=0.0108) (Fig. 6C, 6E). Combinations of Prox-1 and NFATc1 siRNAs did not further decrease VEGFR-3 or podoplanin protein (Fig. 6D, 6E). These data provide evidence that NFATc1 regulates endogenous expression of LEC genes VEGFR-3 and podoplanin, but not Prox-1.
To understand the role of NFATc1 signaling in VEGF-A mediated lymphangiogenesis, HMVEC-L were transfected with NFATc1 and Prox-1 siRNA, treated with VEGF-A and analyzed for VEGFR-3 and podoplanin expression. VEGF-A increased protein levels of VEGFR-3 and podoplanin by 3.2-fold (p=0.0003) and 4.1-fold (p=0.007) respectively (Fig. 7A, 7B). NFATc1 siRNA reduced VEGF-A-induced VEGFR-3 levels by 65% (p=0.004) (Fig. 7C, 7D) and podoplanin levels by 75% (p=0.0063) (Fig. 7C, 7E). Comparable levels of inhibition were achieved with siRNA for Prox-1, which reduced VEGF-A-induced VEGFR-3 levels by 58% (p=0.0078) (Fig. 7C, 7D) and podoplanin levels 70% (p=0.0136) (Fig. 7C, 7E). The combination of NFATc1 and Prox-1 siRNA did not further decrease VEGF-A-induced VEGFR-3 expression than with each siRNA alone (Fig. 7C, 7D). However, in combination, NFATc1 and Prox-1 siRNAs decreased VEGF-A-induced podoplanin levels 3-fold (p=0.0175) better than with siRNA for NFATc1 and 3.8-fold (p=0.0076) better than with siRNA for Prox-1 (Fig. 7C, 7E). There was no change in VEGFR-2 expression by either the individual or the combination of siRNAs (data not shown). These data provide evidence that both NFATc1 and Prox-1 regulate VEGF-A-induced LEC gene expression and that in combination the transcription factors may regulate certain VEGF-A-induced LEC genes.
Co-immunoprecipitation was used to assess whether NFATc1 and Prox-1 physically interact. In lysates from HMVEC-L treated with the calcium activator thapsigargin (200 nM for 2 hours) or VEGF-A-165 (20 ng/ml for 2 hours), NFATc1 antibody pulled down NFATc1 protein along with Prox-1 protein (Fig. 7F). In lysates from untreated control cells (Cntrl, Fig 7F), NFATc1 antibody pulled down only NFATc1, indicating no NFATc1-Prox-1 interaction in nonactivated cells. These data suggest that VEGF-A activates NFATc1 in HMVEC-L and may promote NFATc1/Prox-1 physical interaction.
A constitutively activated NFATc1 (ca-NFATc1), always localized in the nucleus and unaffected by calcineurin activity (Beals et al., 1997), was tested for the ability to activate the luciferase reporters with the proximal promoters of LEC genes, FGFR-3 (McEwen and Ornitz, 1998) and podoplanin (Ramirez et al., 1997) in the mouse endothelial cell line MFLM-91U (Akeson et al., 2000). FGFR-3 (Shin et al., 2006) and podoplanin (Hong et al., 2002) are Prox-1 regulated, lymphatic specific genes. In co-transfection assays, the FGFR-3 luciferase reporter was activated 2.79-fold by (ca)-NFATc1 and podoplanin luciferase reporter activity was increased 1.5-fold (Fig. 8). DSCR(e4) an intragenic element upstream of DSCR exon 4 containing multiple NFAT binding elements (Lange et al., 2004), used to confirm NFATc1 activity, was activated 6-fold (Fig. 8). These data demonstrate that NFATc1 alone activates lymphatic gene expression.
We have addressed the specific role of the transcription factor NFATc1 in lymphatic development. NFATc1 was coexpressed with lymphatic markers Prox-1, VEGFR-3 and podoplanin on the lymphatic endothelium during development as well as on mature lymphatics. Using both in vitro and in vivo analyses, we have shown that calcineurin-NFATc1 signaling plays a role in lymphatic patterning after segregation from the blood vasculature in development and also during injury induced lymphangiogenesis. These results demonstrate the role of calcineurin-NFAT pathway in lymphangiogenesis and suggest that NFATc1 is the principle NFAT involved.
In NFATc1 null mice, endothelial cells sprouted and migrated away from the cardinal vein, but coalesced poorly forming smaller lymph sacs. Like Prox-1 (Wigle et al., 2002; Wigle and Oliver, 1999), NFATc1 is not required to initiate endothelial cell sprouting from the cardinal vein. The failure to form lymph sacs in NFATc1 null embryos cannot be attributed to general vascular defects. As reported by others (de la Pompa et al., 1998; Phoon et al., 2004; Ranger et al., 1998), the morphology of the blood vasculature including cardinal vein appeared normal (Fig. 2). While VEGFR-3 expression on the cardinal vein and the endothelial sprouts is reduced in Prox-1 null mice (Wigle et al., 2002; Wigle and Oliver, 1999), we did not observe a decrease in expression of either VEGFR-3 or podoplanin in NFATc1 null mice at E11.5. NFAT transcription factors often exhibit functional redundancy (Hogan et al., 2003). While we find no expression of NFATc3 by the LEC (Supplemental Fig. 1), it is possible that other NFAT family members compensate for NFATc1 during regulation of VEGFR-3 and podoplanin in early lymphatic development.
In a second approach, we used CsA to inhibit calcineurin activation of NFATs. We selected a window for in utero CsA treatment after its known effects on blood vascular development (Graef et al., 2001) and endocardial valve elongation (Chang et al., 2004). When embryos were treated in utero from E12.5 to E14.5 with CsA, we found irregular LEC sprouts coming from the jugular lymph sac and diminished podoplanin and FGFR-3 expression. These data support a role for calcineurin-NFAT signaling in developmental lymphatic patterning after initial sprouting from the cardinal vein. Further studies are needed to define the role of NFATc1 and other NFAT family members at this stage of lymphatic development.
Podoplanin, a Prox-1 regulated gene (Hong et al., 2002), is a transmembrane glycoprotein that promotes endothelial cell adhesion, migration and tube formation in vitro. Podoplanin null mice have an increased number of non-anastomozing, blind-ended and irregular capillary networks suggestive of impaired lymphatic patterning and also display significant edema (Schacht et al., 2003). We have shown that siRNA against NFATc1 in HMVEC-L (with and without VEGF-A) and in utero inhibition of calcineurin with CsA decreased podoplanin expression. These loss of function analyses suggest that the calcineurin-NFATc1 signaling pathway controls the expression of podoplanin during late lymphatic development, lineage maintenance, as well as following injury. We propose that edema seen in CsA treated embryos may be due to a reduction in podoplanin.
The regulation of VEGFR-3 is more complex. While VEGFR-3 is associated with the lymphatic and not blood endothelium in adult tissues, in early development VEGFR-3 is expressed on blood vasculature as well. VEGFR-3 null mice die at E9.5 from cardiovascular abnormalities (Dumont et al., 1998), even before the development of lymphatic vasculature. VEGFR-3 is also expressed by fenestrated capillaries in organs and angiogenic blood vessels in tumors as well as in inflammation (Baluk and McDonald, 2008). In Prox-1 null mice, there is not a complete loss, but a decrease in VEGFR-3 expression (Wigle et al., 2002). Transcription factors other than NFATc1 and Prox-1 likely control VEGFR-3 expression and may explain the lack of in vitro and in vivo correlation of VEGFR-3 expression in our experiments. Further, the difference in VEGFR-3 and podoplanin regulation suggests that all LEC genes are not regulated in the same way during development and injury.
Neither NFATc1 nor Prox-1 expression is exclusive to the lymphatic endothelium. NFATc1, but not Prox-1, is expressed on endothelial cells undergoing differentiation to cardiac valves, some cardinal vein and jugular vein endothelial cells, (Fig. 1, Supplemental Fig. 1) and osteoclast precursors (Hogan et al., 2003). Prox-1, but not NFATc1, is expressed on the epithelium of the eye (Cui et al., 2004), neuroendocrine cells of lungs (Wigle and Oliver, 1999) and developing hepatic cells (Fig. 1). Our data suggests that NFATc1 and Prox-1 are coexpressed only on LEC and are required for the expression of a subset of LEC genes in development as well as injury–induced lymphangiogenesis. Both NFATs and Prox-1 are weak transcription factors and often require cofactors for optimal transcription induction (Cui et al., 2004; Hogan et al., 2003). NFATs cooperate and act synergistically with homeodomain transcription factors such as TTF-1 (Dave et al., 2004) and Oct-1 (Bert et al., 2000). Although Prox-1 is a homeodomain transcription factor (Oliver et al., 1993) and co-immunoprecipitation studies showed weak NFATc1-Prox-1 physical interaction following culture with VEGF-A, we found no evidence for NFATc1-Prox-1 synergism for gene activation. Other transcription factors acting with NFATc1 and Prox-1 are likely required for full LEC gene regulation.
Sox-18 has recently been shown to induce Prox-1 during developmental lymphangiogenesis (Francois et al., 2008), however the inductive signals for NFATc1 during development remain to be elucidated. The in vitro and in vivo data shown here suggest that VEGF-A may be upstream of NFATc1 in injury-induced lymphangiogenesis. Following VEGF activation only Prox-1 positive cells had increased nuclear NFATc1 in vivo and in vitro. We determined that VEGF-D, VEGF-C and VEGF-C156S, a VEGF-C mutant that activates VEGFR-3 exclusively, did not induce NFATc1 nuclear translocation in Prox-1 positive HMVEC-L, suggesting that activation of VEGFR-2 and not VEGFR-3 induces NFATc1. Lymphangiogenesis requires activation of VEGFR-3 by VEGF-C or VEGF-D (Saharinen et al., 2004). VEGF-C promotes the sprouting of Prox-1 positive LEC precursors cells from the cardinal vein (Karkkainen et al., 2004). We propose that lymphangiogenesis also requires activation of VEGFR-2 for induction of the calcineurin/NFAT pathway.
Earlier studies have proposed a model for the role of NFATc1 during lineage specification of osteoclasts and T-cells. According to the model, NFATc1 directs terminal differentiation of a committed precursor into the fully mature state (Hogan et al., 2003). We propose NFATc1 plays a similar role in regulating developmental or injury induced lymphangiogenesis. In our model, venous endothelial precursors destined to become mature venous endothelial cells become committed to a lymphatic lineage upon the expression of the transcription factor Prox-1. Activation of NFATc1 supports final differentiation of already committed lymphatic precursor into a mature lymphatic endothelial cell. NFATc1 promotes the organization of sprouted lymphatic endothelial cells into lymph sacs in early lymphatic development and controls the expression of VEGFR-3, podoplanin and FGFR-3 in developing and matured lymphatic vessels. The spatial and temporal events regulating NFATc1 expression and activation in embryonic lymphangiogenesis are not known, but our data shows that Prox-1 and NFATc1 are not reciprocally regulated.
Injury-induced lymphangiogenesis follows a different path. We propose that following injury when growth factors including VEGF-A are increased, a subset of venous BEC are reprogrammed to express Prox-1 and NFATc1 leading to lineage reselection as LEC. In this context, NFATc1 and Prox-1 cooperate to induce certain LEC genes. Further studies are required to define the nature of the NFATc1 and Prox-1 interaction, elucidate the cofactors required by NFATc1 and identify the activators of and downstream target genes of NFATc1 in both developmental and injury-induced lymphangiogenesis.
NFATc1 null mice (Ranger et al., 1998) were a gift from Dr. Laurie Glimcher (Harvard School of Public Health, Boston). The VEGF-A inducible bitransgenic mouse system SPCrtTA/tetOVEGF was previously described (Akeson et al., 2003). Mice null for the regulatory subunit of calcineurin (CnAβ null mice) were a gift from Dr. Jeffery Molkentin (CCHRF, Cincinnati) (Bueno et al., 2002a; Bueno et al., 2002b). The CnAβ null mice were crossed with SPCrtTA/tetOVEGF bitransgenic mice to create CnAβ−/−/SPCrtTA/tetOVEGF embryos with inducible VEGF-A and inhibited calcineurin. To induce VEGF-A in the distal lungs, the time-pregnant dams were administered doxycycline in food and water (Akeson et al., 2003). Non-transgenic and single transgenic littermates served as controls. For cyclosporine-A (CsA) treatment, time-pregnant dams were injected intraperitoneally with 40 mg/kg CsA daily in 200 microliter volume from E12.5 to E14.5 and embryos were harvested at E14.5. CsA [Sandimmune (Novartis), 50 mg/ml in cremaphor EL and ethanol 67:33 v/v] was diluted in saline. Vehicle treated control mice received cremaphor EL (Sigma) and ethanol 67:33 v/v diluted in saline. All animal procedures were conducted under the protocols approved by the Institutional Animal Care and Use Committee of Cincinnati Children’s Hospital.
For tissue collection, timed-pregnant dams were euthanized by carbon dioxide inhalation and embryos removed by caesarean section. Embryonic tissue was fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 4 micron for immunohistochemistry (Greenberg et al., 2002). The primary antibodies were: Prox-1 (Fitzgerald industries, 1:300), NFATc1 (Santa Cruz Biotechnologies, 1:30), VEGFR-3 (R&D Systems, 1:500), podoplanin (DSHB, University of Iowa, 1:500) and FGFR-3 (Santa Cruz, 1:60). The secondary antibodies were biotinylated goat anti-mouse IgG1 (Southern Biotech, 1:200) and Alexa 647, Alexa 488 or Alexa 594 conjugated antibodies (Molecular Probes). Alexa-568-conjugated GSL-B4 isolectin (Molecular Probes) with 10 mM CaCl2 and 20 mM MgCl2 was used to identify all endothelial cells (Akeson et al., 2003; Ponder et al., 1985). For immunofluoroscence, serial sections were stained for various lymphatic markers and images captured using the apotome feature of Zeiss Axioscope Microscope (Carl Ziess) that captures pseudoconfocal Z-stack images. For DAB immunohistochemistry, images were captured using the bright field feature of the Zeiss Axioscope Microscope (Carl Ziess). HMVEC-L were treated with VEGF-C, VEGF-C156S, VEGF-D (all from R&D Systems) or VEGF-A-165 (EMD Bioscience) and analyzed for NFATc1 nuclear translocation. For immunohistochemistry HMVEC-L were lifted with 0.02% EDTA (Gibco) and after cytospin, fixed in 1% paraformaldehyde, permeabilized with 0.3 % TritonX-100 (Sigma) in PBS, blocked and stained for Prox-1 and NFATc1. Secondary antibodies were donkey anti-rabbit IgG Alexa 594 and goat anti-mouse IgG1 Alexa 488 (Molecular Probes). Nuclei were detected using Vectashield mount with DAPI (Vector Labs). For each quantification, 3 to 4 independent HMVEC-L cultures were evaluated with 200–300 cells counted/slide.
In a blinded study where the genotypes of SP-CrtTA/tetOVEGF165/CnAβ−/− and control mice were unknown, ten images of 20X magnification per lung section were analyzed for NFATc1, Prox-1 and VEGFR-3 expression using color discrimination protocols of MetaMorph software. Airspaces and open vascular lumens were delineated and the areas subtracted for calculating total lung area (Bolender et al., 1993). The levels of NFATc1, Prox-1 and VEGFR-3 in the lungs were quantified by the ratio of ‘area of signal’/‘total lung area’. Mean, standard error of the mean and the significance were determined using a 2-tailed Student’s t test for n=4–6 mice in each group. For quantifying the luminal area of developing lymph sacs, we used a modification of protocol of Schacht et al. (Schacht et al., 2003; Streit et al., 1999). LEC were indentified by Prox-1 immunostaining and two sets of serial sections with five to six non overlapping fields per section at 10X and 20X magnification were examined and area of the cardinal vein, the number of lymphatic lumens per μm2, the area of individual lymphatic lumens, and the number of Prox-1 positive cells in the lumens and outside lumens were determined. Mean, standard error of the mean and the significance were determined using a 2-tailed Student’s t test for n=4 (wild type and NFATc1 null) and n=3 (NFATc1 heterozygote) mice in each group.
Human microvascular endothelial cells from lung (HMVEC-L) (Lonza) were maintained in EGM-2-MV media (Lonza) at 37°C, 95% humidity and 5% CO2 and used through passage 7. For activation studies, HMVEC-L were cultured overnight in EGM-2MV media with 1% serum and without supplemental VEGF-A and FGF (EGM-1%). For flow cytometry, cells were lifted with 0.02% EDTA (Gibco), washed with phosphate-buffered saline (PBS), blocked and incubated with PE-conjugated mouse anti-human VEGFR-3 (R&D Systems), mouse anti-human podoplanin (Fitzgerald Industries) or isotype controls for 1 hour. Cells were washed in PBS and incubated with goat anti-mouse Alexa 647 secondary antibody for podoplanin (Molecular Probes) for 30 min. Cells were washed and resuspended in 2% paraformaldehyde and analyzed by FACSCalibur flow cytometer (BD Biosciences) and CellQuest software (BD Biosciences). MFLM91U, an immortalized cell line from murine fetal lung mesenchyme (Akeson et al., 2000), were cultured in Ultraculture media (BioWhitaker).
Double stranded Stealth siRNA (25 bp) against NFATc1 and Prox-1 were from Invitrogen. Three siRNA with non-overlapping sequences for both NFATc1 and Prox-1 were used and their sequences are: NFATc1 siRNA set a- Antisense 5′-UUCCGGCACAGUCAAUGACGGCUCG-3′ and Sense 5′-CGAGCCGUCAUUGACUGUGCCGGAA-3′. NFATc1 siRNA set b- Antisense 5′-AGAGAAUUCGGCUUGCACAGGUCCC-3′ and Sense 5′-GGGACCUGUGCAAGCCGAAUUCUCU-3′. NFATc1 siRNA set c- Antisense 5′-AGACGUAGAAACUGACGUGAACGGG-3′ and Sense 5′-CCCGUUCACGUCAGUUUCUACGUCU-3′. Prox-1 siRNA set a- Antisense 5′-AAAUGUUUGCCUUCCGGUUGUAAGG-3′ and Sense 5′-CCUUACAACCGGAAGGCAAACAUUU-3′. Prox-1 siRNA set b- Antisense 5′-AUAUCUUGAAGAUCGCCGCACUCGG-3′ and Sense 5′-CCGAGUGCGGCGAUCUUCAAGAUAU-3′. Prox-1 siRNA set c- Antisense 5′-AUAUCAAACUGGCUCAUAGUAGGCC-3′ and Sense 5′-GGCCUACUAUGAGCCAGUUUGAUAU-3′. Stealth siRNA negative controls having similar GC content as NFATc1 and Prox-1 siRNA, low GC, medium GC and high GC (Cat. Nos. 12935-200, 12935-300 and 12935-400 respectively) were from Invitrogen. The NFATc1 siRNAs encoded sequences specific to NFATc1 without recognition of other NFATs. HMVEC-L were transfected with 500 pmole of siRNA and Lipofectamine 2000 (Invitrogen) using the manufacturers protocol. Some cells were cultured with human VEGF-A-165 at 20 ng/ml (EMD Chemicals). Lysates were prepared after 48 hours with RIPA buffer (0.1M PBS, 1% NP-40 and protease inhibitor cocktail from Roche). For western analysis, lysates were mixed with reducing sample buffer (Invitrogen), heated at 70°C, separated on 4%–12% Bis-Tris gels (Invitrogen) and transferred onto PVDF membranes (Invitrogen). Membranes were blocked with 5% milk in TBST-II (Tris-buffered saline with Triton-X-100) and incubated with primary antibodies overnight, washed in TBST-II, probed with horseradish peroxidaze (HRP) conjugated secondary antibodies and developed with ECL Plus (GE Healthcare). Band intensities were quantified using ImageQuant software (GE Healthcare) and normalized to actin band intensity. The primary antibodies with their dilutions were: Prox-1 (Upstate, 1:2000), NFATc1 (Santa Cruz Biotechnologies, 1:200), VEGFR-3 (Santa Cruz Biotechnologies, 1:500), podoplanin (Fitzgerald Industries, 1:1000), NFATc2 (Santa Cruz Biotechnologies, 1:150), NFATc3 (Santa Cruz Biotechnologies, 1:500), NFATc4 (Abcam, 1:600) and β-actin (Seven Hills, 1:20,000). The secondary antibodies were goat anti-rabbit-HRP for Prox-1, NFATc4 and VEGFR-3 (EMD Chemicals, 1:20,000) and goat anti-mouse-HRP for podoplanin, NFATc1, NFATc2, NFATc3 and actin (EMD Chemicals, 1:20,000).
Expression vectors for constitutively active NFATc1 (ca-NFATc1) (Beals et al., 1997), the 3KB proximal promoter-luciferase reporter construct with intron1 (377bp) of murine FGFR-3 (McEwen and Ornitz, 1998) and the 1.3KB proximal promoter of rat podoplanin (Ramirez et al., 1997) have been described. MFLM-91U cells were transfected with reporter plasmids, ca-NFATc1 expression vector and pRL-TK (Promega) as a control for transfection efficiency using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 hours after transfection and reporter activity was measured according to protocols for the Dual Luciferase Assay System (Promega). The mean, standard error of the mean and significance were determined using 2-tailed Student’s t test for n=9.
HMVEC-L were treated with 20 ng/ml VEGF-A-165 (EMD Chemicals) or 200nM calcium activator thapsigargin (Sigma) for 2 hours and lysates prepared with RIPA buffer. Lysates of 100 μg total protein were incubated with NFATc1 antibody (Santa Cruz Biotechnologies) or mouse IgG1 isotype (Southern Biotech) for 3 hours at 4 °C with rotation. Antigen-antibody complexes were pulled down by adding 20% slurry of Sepharose A/G beads (Pierce) according to the manufacturer’s protocol. The beads were incubated overnight at 4 °C with rotation, washed with binding buffer (Pierce), mixed with reducing sample loading buffer (Invitrogen), subjected to western analysis and probed for NFATc1 and Prox-1 as described earlier.
This research was possible through the excellent technical support from Amanda Herman, Diane Wiginton and Traci Lynch. We would like to acknowledge the initial contributions and discussions with Dr. Bradford Mallory (University of Cincinnati, Cincinnati, USA). We thank Dr. David Ornitz (Washington University, St. Louis, USA) for the FGFR-3 reporter and Dr. Katherine Yutzey (CCHRF, Cincinnati, USA) for the ca-NFATc1, pBJ5 vectors and critical review of the manuscript. Further, we thank Dr. Jeffery Molkentin (CCHRF, Cincinnati, USA) for the CnAβ null mice and Dr. Laurie Glimcher (Harvard School of Public Health, Boston, USA) for NFATc1 null mice. We also thank Dr. Jeffrey Whitsett (CCHRF, Cincinnati, USA) for his generous support. These studies were supported by NIH HL067807 (A.L.A) and The March of Dimes 6-FY07-315 (A.L.A).
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