lowers blood pressure via nitric oxide and prevents hypertension associated with VEGF inhibition

Sarah B. Sunshinea,1, Susan M. Dallabridaa, Ellen Duranda, Nesreen S. Ismaila, Lauren Bazineta, Amy E. Birsnera, Regina Sohnb, Sadakatsu Ikedac, William T. Puc, Matthew H. Kulked, Kashi Javaheriana, David Zurakowskie,f, Judah M. Folkmana,2, and Maria Rupnicka,b

Divisions of aVascular Biology and cCardiology and Departments of eAnesthesia and fSurgery, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115; bDivision of Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and dDepartment of Medical Oncology, Dana-Farber Institute, Harvard Medical School, Boston, MA 02215

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved May 28, 2012 (received for review April 2, 2012) Antiangiogenesis therapy has become a vital part of the arma- control. VEGF stimulates endothelial NO synthase (eNOS), mentarium against cancer. Hypertension is a dose-limiting toxicity resulting in NO production and lower BP (21, 22). Inhibiting for VEGF inhibitors. Thus, there is a pressing need to address the VEGF in animal studies reduces eNOS expression, leading to associated adverse events so these agents can be better used. The vasoconstriction and hypertension (23). In patients, VEGF in- hypertension may be mediated by reduced NO bioavailability fusion causes rapid NO release and hypotension (24). resulting from VEGF inhibition. We proposed that the hyperten- Endostatin (ES), a fragment of XVIII on chromosome sion may be prevented by coadministration with endostatin (ES), 21, is an endogenous inhibitor (25, 26). This 183- an endogenous with antitumor effects amino acid fragment causes tumor regression in a number of shown to increase endothelial NO production in vitro. We de- animal models (27, 28). Although the molecular pathways are not termined that Fc-conjugated ES promoted NO production in fully defined, major effects of ES signaling include inhibition of endothelial and smooth muscle cells. ES also lowered blood endothelial and survival and angiogenesis. In ad- pressure in normotensive mice and prevented hypertension in- dition, ES induces NO release by cultured endothelial cells and duced by anti-VEGF . This effect was associated with relaxation of ex vivo vascular rings (29, 30). Down syndrome higher circulating nitrate levels and was absent in eNOS-knockout patients have an extra copy of chromosome 21 and a negligible mice, implicating a NO-mediated mechanism. Retrospective study incidence of solid tumors (31). Although several genes likely MEDICAL SCIENCES of patients treated with ES in a clinical trial revealed a small but contribute to this cancer protection (32), it is intriguing to note significant reduction in blood pressure, suggesting that the find- that these patients have ES levels 1.6 times higher than those of ings may translate to the clinic. Coadministration of ES with VEGF the general population (33). Further, their BP is lower than age- inhibitors may offer a unique strategy to prevent -related matched controls (34, 35). These data suggested to us that ES may hypertension and enhance antiangiogenic tumor suppression. enhance the antiangiogenic benefits and lessen the hypertensive effects of VEGF inhibition. Such a finding would offer an ap- nhibiting angiogenesis has proven to be effective in treating proach to improve tolerance to VEGF inhibitors, enabling long- Idiseases dependent on new blood vessel growth. In cancer term treatment with reduced risk of cardiovascular adverse events. patients, antiangiogenic agents prolong progression-free survival Here we show that murine Fc-conjugated ES lowers BP in and improve response rates when used in combination with cy- mice via an NO-mediated mechanism and blocks the hyperten- totoxic (1). In and diabetic sive response to anti-VEGF antibodies. Further, we found fi retinopathy these agents reduce vision loss (2, 3). Consequently, a small but signi cant reduction in BP in patients treated with ES fi angiogenesis inhibitors have been approved in 29 countries thus as part of a clinical trial, suggesting that the nding in mice may far (4), and new applications continue to be explored. be translatable. These results support further investigation into VEGF is a potent angiogenesis stimulator clinically estab- antitumor effects of combined therapy. fi fi lished as an ef cacious target for inhibition. The rst Food and Results Drug Administration-approved angiogenesis inhibitor was bev- acizumab (Avastin), a monoclonal anti-VEGF now ES Lowers BP in C57BL/6 Mice. Normotensive C57BL/6 mice were used to treat several types of cancer (colon, lung, renal, breast) treated with ES at various doses and schedules, and BP and ocular neovascularization. Unfortunately, the enthusiasm for responses were compared with vehicle (saline)-treated controls. All mice were injected daily for 5 d with either ES or saline; and other such inhibitors is tempered by the after a 2-d intervening break, the cycle was repeated. A slight emergence of treatment-limiting adverse cardiovascular effects. reduction in BP at the onset of the study was noted in all the Hypertension is the most common dose-limiting toxicity of groups, likely resulting from further acclimation. Thereafter, BP VEGF inhibitors (5–9). Incidence ranges from 15% to 60%, in the control group remained unchanged, whereas BP of the depending on drug- and patient-related factors still being defined ES-treated groups decreased in relation to dose and schedule (10–14). Early and aggressive initiation of antihypertensive ther- (Fig. 1). Mice receiving ES (4 mg/kg) biweekly had the smallest apy can help maintain the treatment schedule (15) and reduce complications (16, 17). However, baseline blood pressures (BP) often are not reestablished (18). Further, it appears that nearly all Author contributions: S.B.S., S.M.D., J.M.F., and M.R. designed research; S.B.S., S.M.D., patients experience some increase in BP, even if not frank hy- E.D., N.S.I., L.B., A.E.B., R.S., S.I., W.T.P., M.H.K., K.J., J.M.F., and M.R. performed re- pertension (19). This finding is concerning, given that changes in search; S.B.S., S.M.D., N.S.I., M.H.K., D.Z., J.M.F., and M.R. analyzed data; and S.B.S. and BP of as little as 5 mm Hg can significantly impact mortality (20). M.R. wrote the paper. As life expectancy for patients maintained on these newer anti- The authors declare no conflict of interest. tumor agents continues to improve, complications from the ac- This article is a PNAS Direct Submission. companying chronic BP elevations will likely accumulate. 1To whom correspondence should be addressed. E-mail: [email protected]. One widely held explanation for angiogenesis inhibitor-asso- 2Deceased January 14, 2008. ciated hypertension is based on the role of VEGF in NO regu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lation. NO is a potent vasodilator that plays a critical role in BP 1073/pnas.1203275109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1203275109 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Murine Fc-ES resulted in a dose-dependent reduction in systolic BP in C57BL/6 mice. The low-dose group received 4 mg/kg, 2 d/wk (on days 0, 3, 7, 10, 14, and 17) and had the smallest decrease in BP. The high-dose group received 4 mg/kg, 5 d/wk (on days 0–4, 7–11, and 14–18) and had the most stable BP reduction. The concentrated-dose group received the cumulative high weekly dose of 20 mg/kg administered in a single concentrated bolus once a week (on days 0, 7, and 14) and had the greatest declines in BP on the days following treatment; BP levels recovered thereafter. *P < 0.05.

BP reductions, with nadirs of ∼10 mm Hg below controls on the day following treatment (e.g., days 1 and 18). BP rebounded to about 5 mm Hg below controls between drug doses. Mice treated with ES (4 mg/kg) for 5 d consecutively exhibited the most stable reduction in BP, to an average of 10 mm Hg below controls, and did not rebound during the 2-d treatment breaks. The last group, which received a cumulative weekly dose of 20 mg/kg as a single, concentrated bolus and had the most sig- nificant decline in BP, to nearly 20 mm Hg below control fol- lowing drug injections. The BP then recovered to baseline before the next dose.

Endogenous and Exogenous ES Increases NO. Physiologic concen- trations of ES have been shown to increase NO production in endothelial cells and to decrease vascular tone in ex vivo studies Fig. 2. Murine Fc-ES administration (4 mg/kg, 5 d/wk) is associated with (A) (29). We found that murine Fc-ES significantly increased nitrite higher circulating nitrate levels (a measure of NO) and (B) lower systolic BP in production in microvascular (MS1) endothelial cells (P = 0.005) C57BL/6 mice as compared with saline-treated controls. (C) Circulating NO (Fig. S1A) and smooth muscle cells (P = 0.0004) (Fig. S1B) levels were higher in ES-overexpressing mice and were lower in collagen compared with the matched vehicle (saline) controls. Elevated XVIII/ES-knockout mice than in wild-type control mice. These findings cor- NO production by these vascular cells in response to ES in vivo related NO levels with endogenous and exogenous ES levels. would lead to lower BP. fi Serum nitrate levels were signi cantly higher in mice treated nearly 130 mm Hg compared with 105 mm Hg typically seen in P A with ES ( = 0.0003) (Fig. 2 ) than in controls, indicating higher wild-type C57BL/6 mice (36). circulating NO levels. Nitrate levels related inversely to systolic The interaction test was significant for both C57BL/6 mice BP, which segregated the ES and control groups (Fig. 2B): Ni- (F = 8.81, P < 0.001) (Fig. 3A) and eNOS-knockout mice (F = trate levels were higher and BP was lower in ES-treated mice 4.06, P = 0.007) (Fig. 3B), indicating that systolic BP showed a than in controls. different profile from baseline through day 7 for animals treated Nitrate levels also were measured in serum samples from ES- with PBS and those treated with ES. For C57BL/6 mice, BP was overexpressing mice and collagen XVII-knockout mice. ES is unchanged with PBS treatment (F = 2.42, P = 0.063) and de- a proteolytic fragment of collagen XVIII and therefore is absent creased significantly with ES (F = 10.17, P < 0.001) on days 2, 5, in the knockout mice. Compared with wild-type controls, nitrate and 7, compared with baseline (all P < 0.001). In contrast, in levels were higher in the ES-overexpressing mice (P = 0.02) and eNOS-knockout mice, ES had no significant effect on BP (F = lower in the collagen XVIII-knockout mice (P = 0.0005) (Fig. 1.92, P = 0.123). There was a small but significant increase in 2C). Collectively, these findings correlate circulating NO levels systolic BP over baseline on day 5 (F = 2.91, P = 0.033) in mice with endogenous and exogenous ES levels. treated with PBS. The findings suggest a role for eNOS in ES- induced BP reductions. ES Does Not Alter BP in eNOS-Knockout Mice. eNOS-knockout mice − − were treated with ES at 4 mg·kg 1·d 1 for 5 d followed by a 2-d ES Prevents Anti-VEGF Antibody-Induced BP Elevations in C57BL/6 break. As has been reported previously, the eNOS-knockout Mice. Normotensive C57BL/6 mice were treated with anti-murine mice were relatively hypertensive, with baseline systolic BP of VEGF antibody, MAb G6-31 (5 mg/kg, 2×/wk on days 1, 4, 8, 11,

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203275109 Sunshine et al. Downloaded by guest on September 24, 2021 Fig. 4. ES prevented the marked BP elevations that accompany VEGF in- hibition. Normotensive mice treated with anti-murine VEGF antibody MAb G6-31 (5 mg/kg on days 1, 4, 8, 11, and 15) developed significant spikes in BP on the day following treatment. Simultaneous ES treatment (4 mg/kg, 5 d/ wk) prevented these BP spikes. Control mice received IgG (5 mg/kg on days 1, 4, 8, 11, and 15) or IgG + ES (4 mg/kg, 5 d/wk). A gradual increase in baseline BP occurred in both control groups and may be related to IgG.

remained unchanged in animals on dual treatment (MAb G6-31 +

ES), resulting in a significant difference between the groups (P = MEDICAL SCIENCES 0.01). Both control groups of mice receiving IgG had reductions in FS on day 47. Although FS was less reduced for mice treated with IgG + ES, the difference between the two groups was not sig- nificant (P = 0.19). Percents of FS on day 47 were similar among the mice treated with MAb G6-31 and both the IgG control groups (IgG alone, P = 0.49; ES + IgG, P = 0.18).

Fig. 3. ES (4 mg/kg, 5d/wk) caused a significant decrease in systolic BP BP Decreases in ES-Treated Patients. BP data were obtained from throughout the treatment period in wild-type C57BL/6 mice (A) but only on a phase II clinical trial of ES in patients with neuroendocrine day 5 in eNOS-knockout mice (B). tumors. The BP data from the first 3 mo of the study (three cycles of ES) were analyzed retrospectively, revealing a small but fi t − P − − signi cant decrease in both systolic BP ( = 2.60, = 0.01) and 15) alone or in combination with ES (4 mg·kg 1·d 1, 5 d/wk) (Fig. 5A) and diastolic BP (t = −2.53, P = 0.012) (Fig. 5B)in (Fig. 4). BP curves in the group treated with MAb G6-31 alone treated patients compared with their pretreatment pressures. On revealed significant spikes on the days following treatment. average, every 10 d on ES was associated with a decrease in These elevations recovered less and less with each subsequent systolic BP of 0.43 mm Hg and a decrease in diastolic BP of 0.26 dose, resulting in a steady increase in baseline BP. ES prevented mm Hg. The confidence intervals are wide, given the variability the BP spikes after MAb G6-31 treatments, although the gradual among patients, although the trend for both BPs is clearly elevation in baseline pressure still occurred. BP in the IgG downward with longer time on ES. control group (5 mg/kg, 2×/wk on days 1, 4, 8, 11, and 15) in- creased slightly above baseline. BP in the IgG + ES control Discussion group did not vary significantly from baseline. The absence of VEGF-mediated angiogenesis is central to tumor progression a BP decrease in the ES group in this study may result from the and has become a therapeutic target for anticancer treatment coadministration of IgG. Correlations between hypertension and (38, 39). Angiogenesis inhibition via anti-VEGF antibodies elevated levels of serum IgG have been reported (37). IgG may (bevacizumab) in combination with chemotherapy has prolonged have contributed to the steady increase in baseline BP in all of progression-free survival and response rates in patients with the groups. advanced breast, colon, and lung (4, 6, 7, 40, 41). This success has directed focus to the side effects of these agents to ES Limits Anti-VEGF Antibody-Induced Cardiosuppression in Mice. enable full and indefinite use. Echocardiography was used to evaluate the cardiac function of Unanticipated cardiovascular adverse events of bevacizumab mice treated with MAb G6-31 with and without ES compared with and other antiangiogenesis agents have been treatment limiting. controls (ES + IgG or IgG alone) on days 40 and 47 of the study The most prevalent of these adverse events is hypertension, above (Table S1). There was no significant difference in chamber which commonly is defined as >140/90 or >150/100 mm Hg dimensions (ventricular chamber and septal and posterior wall in clinical trials (Common Terminology Criteria for Adverse dimensions during systole and diastole) among the groups or over Events v4.0; http://ctep.cancer.gov/protocolDevelopment/elec- time. The percent of fractional shortening (FS), a measure of tronic_applications/ctc.htm) (42). Identifying an agent that can contractility, also was within normal limits and was similar among mitigate the hypertension and augment the antiangiogenic the groups on day 40 (P = 0.99). However, on day 47, FS was effects of VEGF inhibitors would address this pressing clinical reduced in mice receiving MAb G6-31 alone (P = 0.03) but need. ES may be uniquely suited for this purpose, being an

Sunshine et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 induced events associated with eNOS activation (30). Using single-cell measurements, Wenzel et al. (29) showed that ES induced increases in NO production by human and murine en- dothelial cells that were blocked by an eNOS inhibitor. Ex vivo studies showing vessel relaxation suggested that this mechanism might be physiologically relevant. Our in vivo studies support that supposition. In contrast, Urbich et al. (44) demonstrated that ES interfered with VEGF-induced eNOS phosphorylation at Ser-1177; this interference would be expected to reduce NO bioavailability. The dose and duration of ES exposure may be critical in determining the outcome. We then determined whether the hemodynamic effects of ES could be used to combat the hypertension induced by VEGF inhibitors. The mechanisms regulating this adverse event have yet to be elucidated. However, there is mounting evidence that disruption of VEGF-mediated NO bioavailability plays a signifi- cant role. VEGF causes release of NO and up-regulates eNOS RNA and levels in a dose-dependent manner (21). Dis- rupting this pathway with a VEGF inhibitor would be expected to increase vessel tone and elevate BP. ES may neutralize this process via counteracting effects on the same pathway. We found that administration of an anti-VEGF antibody resulted in two superimposed patterns of BP increase in mice (Fig. 4). Transient spikes in BP to ∼125 mm Hg occurred twice weekly, after each treatment, confirming that VEGF inhibition significantly elevates BP. The time course of the spikes relative to drug dosing offers insight into study designs to ensure capture of such findings. Whether this response occurs in patients would be important and easily obtained information. In addition, over the 19-d study there was a gradual increase of ∼8 mm Hg above baseline to just under 120 mm Hg. The two patterns of BP in- crease may indicate multiple effects from VEGF inhibition. For example, NO reduction can cause transient functional (vaso- constriction) changes acutely and persistent structural (e.g., vascular remodeling, rarefaction) changes when chronic. The implication is that the hypertension may be less reversible and more challenging to manage for patients on long-term anti- Fig. 5. ES decreased BP in patients. BP was recorded from 42 patients angiogenic therapy. treated with rhES as part of a phase I clinical trial for neuroendocrine cancer. fi fi Measurements from the first three cycles show a small but significant de- Our ndings are consistent with the ndings of Facemire et al. crease in both systolic BP (A) and diastolic BP (B). Predicted values are in- (23), who demonstrated that mice treated with an anti-VEGFR2 dicated by solid lines; 95% confidence intervals for individual patients are receptor antibody had a 10 mm Hg increase in BP that was indicated by dashed lines. abolished with N-nitro-L-arginine methyl ester. VEGFR2 mod- ulates eNOS production of NO through multiple signaling pathways (45, 46). The hypertension in their study was associated angiogenesis inhibitor shown to increase NO bioavailability with significant reductions in the expression of endothelial and – in vitro (27 30). neuronal NO synthases in the kidney; such reductions would be fi We found that ES signi cantly lowered BP in normotensive expected to reduce NO bioavailability. mice by up to ∼10 mm Hg in a dose- and schedule-dependent Combination treatment with ES and anti-VEGF antibody fashion (Fig. 1). Despite the long drug half-life (weeks), daily completely abolished the dramatic BP spikes seen with the an- administration rather than a single weekly bolus was needed to tibody alone. Small but significant reductions in both systolic and achieve a sustained reduction in BP. This result is in contrast to diastolic BP in 42 patients treated with ES in clinical trials sug- the treatment schedule that is effective for tumor suppression, gests that this mechanism may translate to patients (Fig. 5). The suggesting that a different mechanism may be responsible (43). results from the clinical trial showing a statistically significant A NO-mediated mechanism is supported by our findings that ES decrease in BP from ES treatment, in combination with the increased NO release from endothelial and smooth muscle cells animal studies, suggest that ES is likely sufficient to prevent the in vitro (Fig. S1). These cell types regulate vascular tone and BP. hypertensive spikes induced by anti-VEGF antibody. It is hoped This finding is consistent with previous studies showing that ES that a prospective clinical trial will be carried out to validate induces NO release by cultured endothelial cells and relaxation this finding of ex vivo vascular rings (29, 30). Further, we show that circu- The ES molecule used in prior clinical trials had a short half- lating nitrate levels were higher in ES-treated mice than in life (∼2 h) with variable potency resulting from difficult synthesis controls (Fig. 2). The association between ES and NO in vivo and molecular instability. Development of a recombinant human is strengthened further by our finding that nitrate levels were ES conjugated to the Fc domain of IgG has made such trans- higher in plasma from ES-overexpressing mice and were lower in lation feasible (43). The half-life of Fc-ES is measurable in collagen XVIII-knockout mice than in wild-type animals. weeks, similar to that of bevacizumab. The estimated doses ES did not reduce BP significantly in eNOS-knockout mice needed, based on preclinical studies, are orders of magnitude (Fig. 3), implicating eNOS as a mediator of this hemodynamic lower than the clinically tested drug because of markedly im- effect. This result is consistent with other studies showing that proved potency. The Fc-conjugated form of ES is more suitable short-term exposure of bovine aortic endothelial cells to ES for clinical trials.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203275109 Sunshine et al. Downloaded by guest on September 24, 2021 Antiangiogenic agents have been shown to reduce cardiac throughput, allowing a larger number of mice to be measured within a 4-h function (18) in addition to hypertension. The influence of period. Mice were acclimated to the BP systems over several days (50). VEGF and NO on BP potentially could contribute to the re- Baseline BP then was established for each animal as the average of three sets fi duced left ventricular ejection fraction (47). We found that anti- of readings acquired on separate days with no signi cant difference in the VEGF antibody, MAb G6-31, caused a reduction in fractional sets of measurements for each mouse. shorting after prolonged treatment that was prevented by ES NO Assay in Mice. Serum samples were collected from wild-type mice treated (Fig. 5). The reduction cannot be fully attributed to VEGF in- with saline (control) or ES (4 mg/kg/d, 5×/wk). Samples from untreated col- hibition, however, because it also occurred in control mice fi fl lagen XVIII-knockout mice (51, 52) and ES-overexpressing mice (53) were treated with IgG. This nding may re ect an effect of the generously provided by Sandra Ryeom (Children’s Hospital, Boston). The NO immunoglobulins, which have been implicated in cardiac dys- assay was performed using a quantitative fluorometric extracellular NO as- function in dilated cardiomyopathy patients (48). Although say (Calbiochem) following the manufacturer’s instructions. Plasma was immunoglobulins are presumed to be primarily cardiac auto- thawed, filtered using Ultracel 10-kDa cutoff Microcons (Millipore) by cen- antibodies, the Fc part of IgG molecules may cause negative trifugation (0.5 h, 4 °C), and samples were processed in the NO assay. Plates inotropic effects in cardiac myocytes, as well (49). were read using a Wallac 1420 Multilabel Victor3 microplate reader (Perkin- Our results suggest that ES increases NO production and/or Elmer), with excitation at 355 nm and emission at 430 nm. availability, reduces BP in vivo, and prevents hypertension in- duced by VEGF inhibition. These finding suggest that combi- Retrospective Analysis of BP from Patients Receiving ES. In a multicenter phase nation therapy with these agents may augment the antitumor II clinical trial, patients with advanced neuroendocrine tumors were treated effects while preventing the adverse cardiovascular effects. Such with recombinant human ES (rhES). Forty-two patients were enrolled in the a strategy would be expected to improve patient tolerance to study from Dana-Farber Cancer Institute (Boston), Massachusetts General Hospital (Boston), the Beth Israel Deaconess Medical Center (Boston), or the these highly effective agents and further advance the paradigm University of California, San Francisco Comprehensive Cancer Center (San shift in cancer management toward a chronic disease. Francisco). The treatment schedule was 30 mg/m2 rhES twice a day with a starting total daily dose of 60 mg/m2 daily without treatment breaks. One Materials and Methods cycle was 28 d of treatment. If patients did not reach a steady-state trough Animal Care. All animal procedures were approved by the Animal Care and level of 300 ng/mL, the daily dose was increased to 90 mg/m2 (54). Three ’ Use Committee of Children s Hospital, Boston, and were in compliance with cycles of BP data collected from these patients over 3 mo of continuous fi institutional guidelines. Unless otherwise speci ed, 16-wk-old male C57BL/6J treatment were analyzed retrospectively. mice (Jackson Laboratories) were used. Mice were caged in groups of three Additional methodology is described in SI Materials and Methods.

to five and were fed animal chow and water ad libitum. MEDICAL SCIENCES ACKNOWLEDGMENTS. We thank Dr. Napoleone Ferrara and Dr. Stuart Bun- BP Measurements. BP was measured using noninvasive tail-cuff systems to ting of Genentech for generously contributing MAb G6-31 and their exper- avoid surgical manipulation. The IITC 12M179 12-channel system (IITC Life tise to these studies and Dr. Louis G. D’Alecy, University of Michigan Medical Science, Inc.) was used for the ES dose study (Fig. 1). Thereafter, studies were School, for contributing his expertise in hemodynamic monitoring. We also conducted using three Kent Coda-6 systems (Kent Scientific) to increase thank EntreMed for access to the ES clinical trial BP data.

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