Endothelin-1 and antiangiogenesis.

Antiangiogenesis, targeting vascular endothelial growth factor (VEGF), has become a well-established treatment for patients with cancer. This treatment is associated with nitric oxide (NO) suppression and a dose-dependent activation of the endothelin system, resulting in preeclampsia-like features, particularly hypertension and renal injury. Studies in endothelium NO synthase (eNOS)-deficient mice and pharmacological treatment with endothelin receptor blockers and sildenafil indicate that an activated endothelin system, rather than NO suppression, mediates the side effects of angiogenesis inhibitors. Activation of the endothelin system is also observed in preeclamptic women, where it is related to the increased placental production of sFlt-1, the soluble form of the VEGF receptor-1. This receptor binds VEGF, thereby having the same consequences as antiangiogenic treatment with VEGF inhibitors. The side effects of antiangiogenic treatment in patients with cancer may be dose limiting, thereby impairing its therapeutic potential. In addition, because endothelin exerts proangiogenic effects, investigation of the effects of endothelin receptor blockade in patients with cancer treated with angiogenesis inhibitors is warranted.

Effect of high salt diet on blood pressure and renal damage during vascular endothelial growth factor inhibition with sunitinib.

BACKGROUND: Antiangiogenic treatment with the multitargeted vascular endothelial growth factor (VEGF) receptor inhibitor sunitinib associates with a blood pressure (BP) rise and glomerular renal injury. Recent evidence indicates that VEGF derived from tubular cells is required for maintenance of the peritubular vasculature. In the present study, we focussed on tubular and glomerular pathology induced by sunitinib and explored whether a high salt (HS) diet augments the BP rise and renal abnormalities. METHODS: Normotensive Wistar Kyoto (WKY) rats were exposed to a normal salt (NS) or HS diet for 2 weeks and subsequently for 8 days to sunitinib or vehicle administration after which the rats were euthanized and kidneys excised. Mean arterial pressure (MAP) was telemetrically measured. Urine was sampled for proteinuria and endothelinuria, and blood for measurement of endothelin-1, creatinine and cystatin C. RESULTS: Compared with the NS diet, MAP rapidly rose by 27 ± 3 mmHg with the HS diet. On sunitinib, MAP rose further by 15 ± 1 with the NS and by 23 ± 4 mmHg with the HS diet (P < 0.05). The HS diet itself had no effect on proteinuria, endothelinuria or the plasma levels of endothelin-1, creatinine and cystatin C. Only with the HS diet, sunitinib administration massively increased proteinuria and endothelinuria and these two parameters were related (r = 0.50, P < 0.01). Likewise, renal glomerular pathology was enhanced during sunitinib with the HS diet, whereas tubulointerstitial injury or reduced peritubular capillary density did not occur. CONCLUSIONS: An HS diet induces a marked BP rise in WKY rats and exacerbates both the magnitude of the BP rise and glomerular injury induced by sunitinib.

Cumulative dose of bevacizumab associates with albuminuria rather than podocyturia in cancer patients.

Angiogenesis inhibition with bevacizumab, a monoclonal antibody against vascular endothelial growth factor A (VEGF-A), is an anticancer treatment associated with hypertension and renal glomerular toxicity referred to as a preeclampsia-like syndrome. In preeclampsia, podocyturia predates proteinuria and clinical features of preeclampsia, and is regarded as a biomarker of ongoing glomerular injury. Using a quantitative polymerase chain reaction of the podocyte-specific molecules nephrin, podocin, and VEGF-A in the urine, we examined whether podocyturia is present in bevacizumab-treated cancer patients, and whether it relates to proteinuria and the cumulative dose of bevacizumab. Urine samples were cross-sectionally collected from 43 bevacizumab-treated patients, 21 chemotherapy-treated patients, and 7 healthy controls. Urinary protein-to-creatinine ratio (mean and range) was 32.0 mg/mmol (5.2-284.4) in the bevacizumab group, compared with 11.4 mg/mmol (1.1-21.0) in the chemotherapy group and 7.4 mg/mmol (3.9-16.5) (P < .05) in healthy controls, whereas urinary albumin-to-creatinine ratio values in the three groups were, respectively, 18.9 mg/mmol (0.1-227.7), 1.5 mg/mmol (0.2-3.5), and 0.2 mg/mmol (0.1-0.4) (P < .05). The cumulative dose of bevacizumab ranged from 550 to 93,628 mg. Urinary podocin mRNA expression was undetectable in 59% of participants, urinary nephrin mRNA expression per mmol creatinine ranged from 0.0 to 5.3 and urinary VEGF-A mRNA expression from 0.0 to 2.7. Urinary nephrin mRNA expression did not correlate to the albumin-to-creatinine ratio or the cumulative dose of bevacizumab, whereas the latter correlated with the albumin-to-creatinine ratio (r = 0.77; P < .001). Our results demonstrate that the cumulative dose of bevacizumab is closely correlated with albuminuria but not with podocyturia as measured with the quantitative polymerase chain reaction technique, challenging the feasibility of this measurement to monitor ongoing glomerular injury in patients chronically treated with bevacizumab.

Salt Sensitivity of Angiogenesis Inhibition-Induced Blood Pressure Rise: Role of Interstitial Sodium Accumulation?

In response to salt loading, Na+ and Cl- accumulate in the skin in excess of water, stimulating skin lymphangiogenesis via activation of the mononuclear phagocyte system cell-derived vascular endothelial growth factor-C-vascular endothelial growth factor type 3 receptor signaling pathway. Inhibition of this pathway results in salt-sensitive hypertension. Sunitinib is an antiangiogenic, anticancer agent that blocks all 3 vascular endothelial growth factor receptors and increases blood pressure. We explored the salt dependency of sunitinib-induced hypertension and whether impairment of skin lymphangiogenesis is an underlying mechanism. Normotensive Wistar-Kyoto rats were exposed to a normal or high salt with or without sunitinib administration. Sunitinib induced a 15 mm Hg rise in telemetrically measured blood pressure, which was aggravated by a high-salt diet (HSD), resulting in a decline of the slope of the pressure-natriuresis curve. Without affecting body weight, plasma Na+ concentration or renal function, Na+ and Cl- skin content increased by 31% and 32% with the high salt and by 49% and 50% with the HSD plus sunitinib, whereas skin water increased by 17% and 24%, respectively. Skin mononuclear phagocyte system cell density increased both during sunitinib and a HSD, but no further increment was seen when HSD and sunitinib were combined. HSD increased skin lymphangiogenesis, while sunitinib tended to decrease lymphangiogenesis, both during a normal-salt diet and HSD. We conclude that sunitinib induces hypertension that is aggravated by high salt intake and not accompanied by impaired skin lymphangiogenesis.

Etiology of angiogenesis inhibition-related hypertension.

Angiogenesis inhibition, targeting vascular endothelial growth factor (VEGF) or its receptors, is an established treatment for solid tumors. A common side effect of this treatment is the development of sometimes severe hypertension. This hypertension is associated with a decrease in nitric oxide production, activation of the endothelin-signaling pathway and renin suppression. The mechanism underlying activation of the endothelin-signaling pathway is not fully understood. Both activation of endothelial cells and disinhibition of the VEGF-induced suppression of endothelin production by endothelial cells may be involved. The development of hypertension can be a reason to discontinue the angiogenesis inhibitor, thereby compromising anticancer treatment, but possibly is also a biomarker for a favorable antitumor response.

Association studies suggest a key role for endothelin-1 in the pathogenesis of preeclampsia and the accompanying renin-angiotensin-aldosterone system suppression.

Women with preeclampsia display low renin-angiotensin-aldosterone system activity and a high antiangiogenic state, the latter characterized by high levels of soluble Fms-like tyrosine kinase (sFlt)-1 and reduced placental growth factor levels. To investigate whether renin-angiotensin-aldosterone system suppression in preeclampsia is because of this disturbed angiogenic balance, we measured mean arterial pressure, creatinine, endothelin-1 (ET-1), and renin-angiotensin-aldosterone system components in pregnant women with a high (≥85; n=38) or low (<85; n=65) soluble Fms-like tyrosine kinase-1/placental growth factor ratio. Plasma ET-1 levels were increased in women with a high ratio, whereas their plasma renin activity and plasma concentrations of renin, angiotensinogen, and aldosterone were decreased. Plasma renin activity-aldosterone relationships were identical in both the groups. Multiple regression analysis revealed that plasma renin concentration correlated independently with mean arterial pressure and plasma ET-1. Plasma ET-1 correlated positively with soluble Fms-like tyrosine kinase-1 and negatively with plasma renin concentration, and urinary protein correlated with plasma ET-1 and mean arterial pressure. Despite the lower plasma levels of renin and angiotensinogen in the high-ratio group, their urinary levels of these components were elevated. Correction for albumin revealed that this was because of increased glomerular filtration. Subcutaneous arteries obtained from patients with preeclampsia displayed an enhanced, AT2 receptor-mediated response to angiotensin II. In conclusion, a high antiangiogenic state associates with ET-1 activation, which together with the increased mean arterial pressure may underlie the parallel reductions in renin and aldosterone in preeclampsia. Because ET-1 also was a major determinant of urinary protein, our data reveal a key role for ET-1 in the pathogenesis of preeclampsia. Finally, the enhanced angiotensin responsiveness in preeclampsia involves constrictor AT2 receptors.

Greater Sensitivity of Blood Pressure Than Renal Toxicity to Tyrosine Kinase Receptor Inhibition With Sunitinib.

Hypertension and renal injury are off-target effects of sunitinib, a tyrosine kinase receptor inhibitor used for the treatment of various tumor types. Importantly, these untoward effects are accompanied by activation of the endothelin system. Here, we set up a study to explore the dose dependency of these side effects. Normotensive Wistar Kyoto rats were exposed to 3 different doses of sunitinib or vehicle. After 8 days, rats were euthanized. Telemetrically measured blood pressure rose dose dependently, from 13 to 30 mm Hg. Proteinuria was present at all doses, but a rise in cystatin C occurred only at the intermediate and high doses. Compared with vehicle circulating endothelin-1 increased dose dependently, whereas 24-hour urinary endothelin excretion decreased. Light and electron microscopy revealed glomerular endotheliosis and ischemia with the intermediate and high doses of sunitinib but completely absent histological abnormalities with the low dose. Podocyte number per glomerular circumference did not change. Glomerular nephrin, Neph1, podocin, and endothelin-converting enzyme gene expression were downregulated in a dose-dependent manner. We conclude that the sunitinib-induced rise in blood pressure requires lower doses than its induction of renal function impairment and that functional changes in glomerular filtration barrier contribute to the occurrence of proteinuria, given the lack of histopathologic changes with the low dose of sunitinib.

Hypertension during vascular endothelial growth factor inhibition: focus on nitric oxide, endothelin-1, and oxidative stress.

SIGNIFICANCE: Angiogenesis inhibition with humanized antibodies targeting vascular endothelial growth factor (VEGF) or orally active small tyrosine kinase inhibitors targeting VEGF receptors has become an established treatment modality for various forms of cancer. A common side effect of angiogenesis inhibition is the development of sometimes severe hypertension, which simultaneously appears to be predictive for a favorable antitumor response. RECENT ADVANCES: Since VEGF increases the expression and activity of endothelial nitric oxide synthase, it has been assumed that the mean blood pressure (MAP) rise during angiogenesis inhibition is caused by a decrease in nitric oxide bioavailability. Yet, the results from experimental and clinical studies exploring this possibility are conflicting. Recent studies provided evidence that the MAP rise during angiogenesis inhibition rather is mediated by activation of the endothelin-1 (ET-1) axis, which, among others, induces oxidative stress. Nevertheless, conclusive evidence for the involvement of reactive oxygen species in the MAP rise could not be obtained so far. CRITICAL ISSUES: The mechanism underlying activation of the ET-1 axis during angiogenesis inhibition is unclear, and this activation was not anticipated in view of studies showing that VEGF stimulates both the expression and production of ET-1 by endothelial cells. FUTURE DIRECTIONS: In fact, this activation of the ET-1 axis may support the use of ET receptor antagonists for the treatment of angiogenesis inhibition-induced hypertension, especially because ET receptor stimulation in vascular smooth muscle cells results in VEGF production and mitogenesis in a mitogen-activated protein kinase pathway-dependent manner.

Treatment of hypertension and renal injury induced by the angiogenesis inhibitor sunitinib: preclinical study.

Common adverse effects of angiogenesis inhibition are hypertension and renal injury. To determine the most optimal way to prevent these adverse effects and to explore their interdependency, the following drugs were investigated in unrestrained Wistar Kyoto rats exposed to the angiogenesis inhibitor sunitinib: the dual endothelin receptor antagonist macitentan; the calcium channel blocker amlodipine; the angiotensin-converting enzyme inhibitor captopril; and the phosphodiesterase type 5 inhibitor sildenafil. Mean arterial pressure was monitored telemetrically. After 8 days, rats were euthanized and blood samples and kidneys were collected. In addition, 24-hour urine samples were collected. After sunitinib start, mean arterial pressure increased rapidly by ≈30 mm Hg. Coadministration of macitentan or amlodipine largely prevented this rise, whereas captopril or sildenafil did not. Macitentan, captopril, and sildenafil diminished the sunitinib-induced proteinuria and endothelinuria and glomerular intraepithelial protein deposition, whereas amlodipine did not. Changes in proteinuria and endothelinuria were unrelated. We conclude that in our experimental model, dual endothelin receptor antagonism and calcium channel blockade are suitable to prevent angiogenesis inhibition-induced hypertension, whereas dual endothelin receptor antagonism, angiotensin-converting enzyme inhibitor, and phosphodiesterase type 5 inhibition can prevent angiogenesis inhibition-induced proteinuria. Moreover, the variable response of hypertension and renal injury to different antihypertensive agents suggests that these side effects are, at least in part, unrelated.

Mechanism of hypertension and proteinuria during angiogenesis inhibition: evolving role of endothelin-1.

Angiogenesis inhibition by blocking vascular endothelial growth factor (VEGF)-mediated signalling with monoclonal antibodies or tyrosine kinase inhibitors has become an established treatment of various forms of cancer. This treatment is frequently associated with the development of hypertension and proteinuria. As VEGF increases the expression and the activity of nitric oxide synthase in endothelial cells, a decrease in the bioavailability of nitric oxide has been proposed as a key mechanism leading to hypertension during angiogenesis inhibition. However, results of clinical and experimental studies exploring this possibility are conflicting. Rarefaction, that is a structural decrease of microcirculatory vessels, has been reported during antiangiogenic treatment, but evidence that it plays a role in development of hypertension is lacking. Elevated circulating and urinary levels of endothelin-1 have been observed in clinical and experimental studies with angiogenesis inhibitors. Furthermore, the observation that endothelin receptor blockers can prevent or revert the rise in blood pressure during angiogenesis inhibition and attenuate proteinuria provides strong evidence that an activated endothelin-signalling pathway is a final common mediator of angiogenesis inhibition-induced rise in blood pressure and renal toxicity.

Impaired vascular contractility and aortic wall degeneration in fibulin-4 deficient mice: effect of angiotensin II type 1 (AT1) receptor blockade.

Medial degeneration is a key feature of aneurysm disease and aortic dissection. In a murine aneurysm model we investigated the structural and functional characteristics of aortic wall degeneration in adult fibulin-4 deficient mice and the potential therapeutic role of the angiotensin (Ang) II type 1 (AT(1)) receptor antagonist losartan in preventing aortic media degeneration. Adult mice with 2-fold (heterozygous Fibulin-4(+/R)) and 4-fold (homozygous Fibulin-4(R/R)) reduced expression of fibulin-4 displayed the histological features of cystic media degeneration as found in patients with aneurysm or dissection, including elastin fiber fragmentation, loss of smooth muscle cells, and deposition of ground substance in the extracellular matrix of the aortic media. The aortic contractile capacity, determined by isometric force measurements, was diminished, and was associated with dysregulation of contractile genes as shown by aortic transcriptome analysis. These structural and functional alterations were accompanied by upregulation of TGF-ß signaling in aortas from fibulin-4 deficient mice, as identified by genome-scaled network analysis as well as by immunohistochemical staining for phosphorylated Smad2, an intracellular mediator of TGF-ß. Tissue levels of Ang II, a regulator of TGF-ß signaling, were increased. Prenatal treatment with the AT(1) receptor antagonist losartan, which blunts TGF-ß signaling, prevented elastic fiber fragmentation in the aortic media of newborn Fibulin-4(R/R) mice. Postnatal losartan treatment reduced haemodynamic stress and improved lifespan of homozygous knockdown fibulin-4 animals, but did not affect aortic vessel wall structure. In conclusion, the AT(1) receptor blocker losartan can prevent aortic media degeneration in a non-Marfan syndrome aneurysm mouse model. In established aortic aneurysms, losartan does not affect aortic architecture, but does improve survival. These findings may extend the potential therapeutic application of inhibitors of the renin-angiotensin system to the preventive treatment of aneurysm disease.