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. Author manuscript; available in PMC: 2020 Nov 19.
Published in final edited form as: Biochem Biophys Res Commun. 2019 Sep 18;519(4):667–673. doi: 10.1016/j.bbrc.2019.09.043

CALCINEURIN INHIBITORS AUGMENT ENDOTHELIAL-TO-MESENCHYMAL TRANSITION BY ENHANCING PROLIFERATION IN ASSOCIATION WITH CYTOKINE-MEDIATED ACTIVATION

Craig B Woda 1,2, Sarah Bruneau 1,2, Anne Linde Mak 1,2, Zdenka Haskova 1, Kaifeng Liu 1, Chandra C Ghosh 1,2,*, David M Briscoe 1,2,*
PMCID: PMC7119266  NIHMSID: NIHMS1543117  PMID: 31542230

Abstract

Calcineurin Inhibitors (CNIs) are routinely used for immunosuppression following solid organ transplantation. However, the prolonged use of these agents leads to organ fibrosis, which limits their efficacy. They also induce TGFβ expression, which is reported to augment endothelial to mesenchymal transition (EndMT), but the role of CNIs in this process is not known. In these studies, we find that FK506 and CsA are potent to increase endothelial cell (EC) proliferation using established in vitro assays (P<0.05). Furthermore, using phosphokinase arrays, we find that each CNI activates the MAPK and Akt/mTOR signaling pathways, and that pharmacological inhibition of each pathway targets CNI-induced proliferative responses (P<0.001). EndMT was evaluated by FACS for N-cadherin and CD31 expression and by qPCR for the expression of α-smooth muscle actin, N-cadherin and Snail. We find that CNIs do not directly induce the dedifferentiation, while TGFβ and hypoxia induce EndMT in small numbers of EC. In contrast, the treatment of EC with the inflammatory cytokine TNFα was potent to elicit an EndMT response, and its effects were most notably in EC following proliferation/doubling. Taken together, these observations suggest that CNIs elicit proliferative responses, which enhance EndMT in association with local inflammation. The clinical implications of these findings are that anti-proliferative therapeutics have high potential to target the initiation of this response.

Keywords: Endothelial Cells, Endothelial-to-Mesenchymal Transition, Calcineurin inhibitors, Tumor Necrosis Factor, Transplantation, Chronic Allograft Rejection

1. Introduction

Microvascular injury is a characteristic component of the alloimmune response, and the degree of endothelial cell loss within an allograft is predictive of subsequent decline in function and the development of chronic rejection [1,2,3]. Although loss of EC within allografts may occur through the process of endothelial to mesenchymal transition (EndMT, [1,4,5,6,7]), the mechanisms underlying the initiation and evolution of EndMT within allografts in vivo are poorly understood. We wished to identify mechanisms that prime EC for EndMT and evaluate whether calcineurin inhibitor post-transplant therapeutics contribute to this process.

EndMT is characterized phenotypically by the de-differentiation of endothelial cells (EC) into fibroblast-like cells [8,9,10,11] including decreased expression of CD31 and VE-cadherin (VE-Cad) and a coincident increase in the expression of fibroblast markers, including α-smooth muscle actin (α-SMA), N-Cadherin (N-Cad) and the transcription factor Snail [8,9]. It is reported to be dependent on TGFβ-elicited signaling in vitro in cultured EC [4,5,8,9] suggesting that CNIs may augment EndMT via their well-established ability to induce TGFβ expression [12,13]. Understanding their effects on EndMT has profound implications for the optimal use of CNIs as therapeutics while avoiding their pro-fibrotic off-target effects.

In these studies, we failed to find any direct effect of the cyclosporine (CsA) or FK506 on the dedifferentiation of EC, but we observed that they are potent to promote EC proliferation and an angiogenesis response. Surprisingly, the treatment of our EC with TGFβ had minimal effects on the development of EndMT, but it was notable following treatment with the pro-inflammatory cytokine cytokine TNFα. Furthermore, we find that EndMT occurs to a greater degree in EC that have undergone multiple doublings. Collectively, these findings indicate that EndMT may occur in EC in association with cytokine-mediated activation, and to a greater extent within EC that have undergone proliferation. Thus, by stimulating cell proliferation, the calcineurin inhibitors CsA and FK506 enhance susceptibility for EndMT in the presence of local intragraft inflammation.

2. Materials And Methods

2.1. Cell culture

Primary cultures HUVECs were isolated from human umbilical cords and cultured in complete endothelial cell growth medium (M-199 media with Pen-Strep, L-Glutamine, 18% FBS, and ECGS) as previously described [14]. For proliferative and signaling experiments, HUVEC were used at sub-passage 3–4 (~60 cellular doublings). For EndMT studies, HUVEC were used at different replicative doublings, those that had undergone <20 doublings and those that had undergone ~80–120 doublings. For hypoxia assays, the cells were grown in a hypoxia chamber (10% air) for 7–10 days. and media changed every 48 hrs.

2.2. Cell proliferation assays

HUVECs were seeded onto 96-well flat bottom cell culture plates (5 × 103 cells per well) in media containing ECGS and 2.5% FBS (starvation media). Cultures were treated with FK506, CsA, or VEGF-A for 72 hrs as indicated in each experiment and/or in the presence of the U0126, rapamycin or neutralizing anti-human VEGF antibody. Proliferation was assessed by measuring 3[H]-Thymidine incorporation (1 μCi/well added during the final 18 hours of culture) in a Tomtec automated cell harvester (Hamden, CT). In all experiments, treatment groups were run in triplicate.

2.3. Sprouting assay

HUVEC (at 40–60 divisions) were trypsinized, allowed to aggregate in hanging drops (750 cells per drop/spheroid) for 24 hours, and embedded into a collagen type I matrix, as described [15]. Spheroids were either untreated, or treated with either FK506 or CsA (0.01–1 mg/ml) or with VEGF-A (as a positive control). After 18hrs, the spheroids were fixed for 15 min with 4% paraformaldehyde and permeabilized for 5 min with 0.2% Triton X-100, and F-actin was visualized using Alexa Fluor® 488-conjugated phalloidin (Invitrogen). Samples were examined by confocal microscopy.

2.4. Real-time PCR

Total RNA was isolated from HUVECs using the RNeasy Isolation kit (Qiagen, Maryland) and cDNA was generated from the mRNA template by qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD), according to the manufacturer’s instructions. Human α-SMA, N-cadherin, Snail, and GAPDH mRNA expression were quantified using the 7300 real-time PCR system and commercially available validated TaqMan® Gene expression primers (Applied Biosystems, Foster City, CA). The relative expression of each gene was compared to GAPDH and calculated according to the 2−ΔΔCt method, as described [16].

2.5. In vivo CNI angiogenesis assay

Chinese Hamster Ovary (CHO) cells, stably transfected to secrete human VEGF (CHO-VEGF) were injected intradermally (1X105 in 20 μl sterile PBS containing 0.5%-BSA) into the ears of nude mice (Taconic laboratories) as described [17]. AA8 cells (Clontech Laboratories, Palo Alto, CA) were used as negative controls. Following injection, the mice were treated daily with FK506 (0.1–1 mg/kg) or CsA (0.3–20 mg/kg) by intraperitoneal injection. Studies were also performed in the presence of either daily intraperitoneal injections of the mTOR inhibitor rapamycin (0.3–1.5mg/kg). Photographs of the ear were obtained on day 4 following injections and the degree of angiogenesis was assessed against mice injected with AA8 cells. Ears were harvested, 10% formalin fixed and paraffin embedded or snap frozen in liquid N2 at the end of the study and processed for histochemistry.

2.6. Statistics

Statistical analyses were performed using the Student t test to compare two groups of data or by one-way ANOVA for the comparison of three or more groups using GraphPad Prism version 6 (GraphPad software). P values <0.05 were considered statistically significant.

3. Results

3.1. Proliferation of Endothelial cells in response to CsA and FK506

We initially treated EC with CsA (0.01 or 0.1 μg/ml) of FK506 (0.01 or 0.1 μg/ml), and we evaluated proliferation using standard 3[H]-Thymidine incorporation. As illustrated in Fig. 1A, we find a significant proliferative response following treatment with either agent (P<0.05 and P<0.001 respectively) as compared to untreated cells. Using the in vitro spheroid sprouting assay (Fig. 1B), we find that each CNI induces a marked angiogenesis response and that CNI-inducible angiogenesis is comparable to that observed following the treatment of EC with VEGF-A (5–20ng/ml, P<0.05).

Figure 1: Calcineurin inhibitors stimulate angiogenesis via VEGF-VEGFR interactions.

Figure 1:

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A) EC (5 ×103 cells per well) were cultured in the absence or presence of FK506 or CsA, and proliferation was evaluated by 3[H]-Thymidine incorporation after 72 hours. Treatment with VEGF-A served as a positive control. Bar graphs represent the fold change in proliferation (mean cpm± SEM) of 12 experiments run in triplicate (*P<0.05; **P<0.01). B) EC were aggregated into spheroids and embedded in collagen matrix in the absence/presence of FK506, CsA or VEGF-A as described in Methods. Photomicrographs are representative of ~ 5 spheroids per condition in n=3 independent experiments. C) EC were cultured with FK506 or CsA in the absence/presence of anti-VEGF-A. Bar graphs represent the fold change in proliferation (mean cpm ± SEM of n≥3 experiments). *P<0.05 and **P<0.01 (within each group). D) EC were cultured with FK506 or CsA for 30 mins and pY1175VEGFR2 and total VEGFR2 expression was evaluated by Western blot. Numbers below each blot represent the densitometric analysis of the ratio of phoshoprotein to loading control (of n≥3 experiments). E) CHO-AA8 or CHO-VEGF cells (1 × 105) were injected subcutaneously into the ears of nude mice that received FK506 (1 mg/kg) or CsA (3 mg/kg) by daily i.p. injection. Photographs were taken on day 4. F) Immunohistochemistry using anti-CD31 of day 4 injected ears from Panel E. The images in panels E and F are representative of n=3 mice.

To investigate whether the CNI-mediated angiogenesis response is VEGF-VEGFR2 dependent, we treated primary cultures of EC with either CsA or FK506 (0.01μg/ml) in the absence or presence of a blocking anti-human VEGF-A antibody. As illustrated in Fig. 1C, we find that anti-VEGF reduced the proliferation of EC under basal conditions as well as following treatment with either CsA or FK506 (P<0.0001). Since CNIs increase VEGF-A mRNA expression ([18,19] and data not shown), we cocultured EC with each CNI and we evaluated the phosphorylation of VEGFR2Tyr1178 by Western blot analysis. As illustrated in Fig. 1D, both CsA and FK506 increased the level of pVEGFR2 at both low (0.01μg/ml) and high (0.1 μg/ml) concentrations.

To test this if CNI-induced angiogenesis is of functional significance in vivo, we used an established model [17] in which VEGF-secreting CHO cells are injected intradermally into the ears of nude mice. The response was determined macroscopically as well as by immunohistochemistry following treatment of mice with FK506 or CsA (by daily by ip injection) vs. untreated controls. Intradermal injection of CHO cells (AA8 cells) that do not secrete VEGF served as additional controls. As illustrated in Figs. 1E–F, the VEGF-induced angiogenesis response was enhanced in mice treated with FK506 or CsA over a 4 day period, although this increase was not statistically significant.

3.2. CNI-mediated proliferation of EC is dependent on MAPK and Akt/mTOR signaling

We next performed a phosphokinase protein array to determine the signaling pathways that are activated in EC following treatment with each CNI. As illustrated in Fig. 2A, we find marked increases in the expression of multiple kinases associated with both the MAPK and the Akt/mTOR signaling pathways. As shown in Figs. 2B–C, we validated these findings by Western blot analysis and find CNI-inducible increases the phosphorylation of ERKT202/Y204 and P70S6KT389 (~2.6 and ~1.9 fold respectively; P<0.01). In addition, the treatment of EC with the pharmacological MAPK inhibitor U0126 (1μM, Fig. 2D) or the mTOR inhibitor rapamycin (Fig. 2E) prior to treatment with either CNI reduced EC proliferative responses by >50% (P<0.01 and P<0.001 respectively). Taken together, these findings are suggestive that CNI-inducible EC proliferation is in part associated with activation of the MAPK and mTOR signaling pathways.

Figure 2: Calcineurin inhibitors induce MAPK and Akt/mTOR signaling in EC.

Figure 2:

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Confluent EC (40–60 doublings) were placed in media containing 2.5% FBS for 6 hours and subsequently treated with FK506 or CsA for 30 minutes. A) Phosphokinase protein arrays analyzing 46 kinases were performed on cell lysates from untreated or treated cells (representative of n=2). B-C) Western blot analysis using anti-pERK1/2 or anti-p70S6K, and total ERK/2 and GAPDH respectively as loading controls (representative of n=4). Numbers below each blot represent densitometric analysis of the ratio of phoshoprotein to loading control. D-E) 72hr proliferation of EC (fold change in mean cpm ± SEM) following treatment with FK506 or CsA in the absence/presence of D) U0126 or E) rapamycin. *P<0.001 within each group of n=7 individual experiments.

3.3. EndMT is a rare event in primary cultures of human EC but is inducible following TNFα-activation

In multiple studies, by FACS and by qPCR, we failed to find that large numbers of our EC undergo dedifferentiation following treatment with either TGFβ1 or TGFβ2 (Fig. 3A). This finding contrasts the previously reported effects of TGFβ-TGFβR interactions on the induction of EndMT in commercially available EC lines [8,9,20]. We next questioned whether EndMT predominantly occurs in EC that have undergone significant doubling/replication by comparing phenotypic changes in later passage EC (80–120 doublings) vs. early passage EC (<20 doublings). Our findings consistently demonstrate that TGFβ1 or TGFβ2 induce only a small (but significant) degree of EndMT after later passages (Fig. 3 A).

Figure 3: Cell doubling augments the dedifferentation of EC and TNFα-induced EndMT.

Figure 3:

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A) Confluent cultures of EC with <20 or 80–120 doublings were treated with TGFβ1 (10ng/ml) or TGFβ2 (2.5ng/ml) alone or in combination with TNFα (100 U/ml) for 7 days. Upper Graph: CD31 and N-cadherin (Ncad) co-expression by FACS. * P<0.001 (compared to untreated cells within each doubling group); Lower Graphs. mRNA expression of α-SMA, Ncad and Snail by qPCR. Bar graphs represent the mean ± SEM of n ≥ 4 experiments for each condition (* P<0.01 compared to untreated cells within each doubling group) B) EC were placed in 21% oxygen (Normoxia) or 10% oxygen (Hypoxia) for 7 days. EndMT was assessed by CD31 and Ncad co-expression by FACS. Bar graphs represent the mean ± SEM of n = 3 experiments. *P<0.05. C) EC were treated with FK506 or CsA for 7 days. EndMT was assessed by CD31 and Ncad co-expression by FACS (upper graph) or by Snail mRNA expression by qPCR (lower graph) in a mean ± SEM of n ≥ 3 experiments. D) EC were treated with FK506 or CsA in the absence/presence of TNFα (100U) for 7 days. EndMT was assessed by CD31 and Ncad co-expression in a mean ± SEM of n ≥ 3 experiments. *P<0.01 compared to untreated cells within each doubling group.

Since EndMT has been reported to occur in vitro following cytokine-induced activation [21,22], we also evaluated dedifferentation and phenotypic EndMT in EC following treatment with TNFα. For these experiments (see Fig.3A), we treated our EC with TNFα alone (100 U/ml) or TNFα in combination with TGFβ1 (10 ng/ml) or TGFβ2 (2.5 ng/ml) using both early passage (<20 doublings) and later passage EC (80–120 doublings). We find a notable EndMT response following treatment with TNFα, in as much as 20 ± 2% of early passage EC (<20 doublings) and 39 ± 5% of later passage EC (80–120 doublings) showed phenoptypic evidence of differentiation (P<0.0001). Furthermore, co-treatment of EC with TNFα and TGFβ1 or TGFβ2 did not have an additive effect on the number of cells undergoing EndMT in either early or in later passage cells. Dedifferentiation was notable by both FACS (CD31 and N-Cad co-expression, Fig 3A, upper panel) as well as by qPCR (α-SMA, N-cad and Snail co-expression; Fig. 3A, lower panels, P<0.05).

TNFα is well established to induce the MAPK pathway, which may also function in fibrosis [5,11,23]. Consistent with these reports, in preliminary experiments we also find that EC fail to undergo a TNFα-inducible dedifferentiation response in the presence of U0126 (data not shown). Thus, cytokine-induced responses in EC may prime for dedifferentiation and EndMT through signals that also regulate cell proliferation (Fig.2 D–E).

3.4. Local hypoxia augments EndMT in EC that have undergone proliferation

Local tissue hypoxia is characteristically associated with chronic allograft rejection [24]. We thus postulated that the collective effects of hypoxia and cytokine-induced activation may drive the dedifferentiation of EC in vivo. As shown in Fig. 3B, we find that hypoxia alone (10% O2 for 7–10 days) had a small but significant effect on EndMT in early passage EC with < 20 doublings (5.2 ± 0.40 vs. 2.16 ± 0.80, P<0.05) and in later passage EC with 80–120 doublings (10.23 ± 1.7 vs. 2.9 ± 0.69, P<0.05). However, the number of EC undergoing EndMT was markedly less than that observed following treatment with TNFα (Fig. 3A), and there was no additive effect of hypoxia on the TNFα-inducible response (data not shown). These findings suggest that hypoxia alone is not a major stimulus for EndMT, and rather that pro-inflammatory cytokines and/or events that elicit EC proliferation prime for subsequent dedifferentiation.

3.5. CNIs do not directly induce an EndMT response

As shown in Fig. 3C, the treatment of EC with either CsA or FK506 has no effect on the number of cells undergoing EndMT (~3–5% cells under all conditions). We next evaluated the effect of each CNI on TNFα-induced EndMT in cells that had, or had not undergone proliferation/cell doubling (Fig 3D). We observed EndMT was present in ~3% of early passage EC (<20 doublings) at baseline and it increased to ~19% in TNFα only. Nevertheless, in EC that had undergone replication (80–120 doublings), EndMT increased from ~6% at baseline to ~36% cells (TNFα only). However, under both conditions there was no additive effect of either CsA or FK506 on the response (Fig 3D). These findings indicate that intracellular signals that elicit cell proliferation are distinct from those that elicit EndMT. Furthermore they indicate that CNIs enhance susceptibility for cytokine-induced EndMT via effects and signals that mediate cell proliferation/cell doubling.

4. Discussion

Calcineurin inhibitors are the cornerstone of immunosuppressive therapy for transplant recipients and patients with select autoimmune diseases [25,26]. However, their use is associated with the development of fibrosis within tissues, especially in renal allografts, which has limited their long-term efficacy following transplantation [26]. In this study, we initially speculated that CNIs induce EndMT in part via their known effects to induce TGFβ [12,13], and thus via TGFβ-regulated dedifferentiation of EC. However, we find that CNIs fail to induce dedifferentiation of EC and, contrary to previous publications [4,5,8,9,27,28], treatment with TGFβ is a weak inducer of EndMT in primary cultures of EC. Rather, we observed that CNIs induce significant proliferation of EC and an angiogenesis response that is in part dependent on VEGFR-induced signaling.

Consistent with other reports [21,22], we observed that EndMT is induced in our EC following treatment with the pro-inflammatory cytokine TNFα, and we additionally find a greater EndMT response in cells that have undergone proliferation (>80 doublings vs. early passage cells). This finding is suggestive that EC may dedifferentiate and undergo EndMT within allografts in association with pro-inflammatory events (e.g. delayed graft function/ATN, rejection and/or humoral injury) but to a greater extent once EC proliferate. Indeed, EndMT has been observed in association with these post-transplantation disease processes [6,7] where local tissue hypoxia may stimulate VEGF-A production and EC turnover [1,24]. These findings are suggestive that ongoing leukocyte-induced injury events that also stimulate EC proliferation/angiogenesis will promote enhanced dedifferentiation and EndMT in association with pro-inflammatory. Our findings also suggest that CNIs may further augment this response by stimulating VEGF-A- and/or VEGFR-dependent proliferation of EC. Thus, intragraft inflammation is the local stimulus that initiates and sustains EndMT, and CNIs likely augment this process by stimulating EC turnover/proliferation.

Our findings are different than some previously published reports [8,9,20] in that we find that EndMT is a rare event in our primary cultures of EC, and TGFβ has minimal effects on dedifferentiation and mesenchymal transition. These observations are consistent with the debate about whether EndMT is an in vitro observation in select cell lineages, and whether it is of relevance in disease processes in vivo [28,29]. Our studies indicate that the number of cell doublings alters EndMT responsiveness and suggest that the dedifferentation of EC in vivo likely requires priming through inflammation, cytokine-induced signaling and/or leukocyte-induced angiogenesis [30]. Of interest, our findings also implicate the MAPK and the mTOR/Akt-induced signaling in CNI-induced activation and proliferation of EC, which in turn enhances susceptibility for dedifferentiation and EndMT [5,11,23,31]. These data are suggestive that the inhibition of these signaling pathways have clinical implications to target both the initiation and evolution of EndMT within allografts in high risk transplant recipients and/or in patients with other diseases where CNIs are used as therapeutics.

In summary, we show that CNIs induce EC proliferative responses but do not directly induce EndMT. Rather we find that TNFα-induced inflammation is a major driver of the dedifferentiation of EC and that EndMT is readily inducible in cells that have undergone cell proliferation/doubling. These findings collectively indicate that CNIs augment susceptibility for dedifferentiation and EndMT within an inflammatory microenvironment. They also provide insight into the molecular basis for the association between CNI use and the development of fibrosis within allografts following transplantation.

Supplementary Material

1

Highlights.

  • Calcineurin inhibitors stimulate endothelial cell proliferation and angiogenesis.

  • The inflammatory cytokine TNFα is potent to elicit an EndMT response.

  • Endothelial cell doubling markedly increases susceptibility to TNFα-induced EndMT.

Acknowledgments

The authors wish to thank past members of the laboratory for initiating these studies, Drs. Kaifeng Liu and Gary Visner for ongoing murine surgical support and Dr. David Milstone for support with hypoxia studies. We also thank Kay Case of the Vascular Biology Division, Brigham and Women’s Hospital for support in the isolation of primary cultures of HUVEC.

Funding: This work was supported by NIH grants T32DK007726 (to CBW and CG) and 4R01AI092305 (to DMB), and by an Independent Investigator Grant from Astellas (to DMB).

Conflict-of-interest disclosure:

All potential conflicts of interest are reviewed and managed by the Office of General Counsel at Boston Children’s Hospital. D.M.B. received grant support from Astellas for these studies and serves as a consultant to aTyr Pharma in an unrelated area.

Footnotes

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NIHMS1543117-supplement-1.pdf (1.2MB, pdf)

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