Chronic hypertension is one of the most important modifiable

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1 Renin Angiotensin System Epidermal Growth Factor Receptor Is Critical For Angiotensin II Mediated Hypertrophy in Cerebral Arterioles Siu-Lung Chan, Shaikamjad Umesalma, Gary L. Baumbach Abstract Angiotensin II (Ang II) is a major determinant of vascular remodeling in the cerebral circulation during chronic hypertension, which is an important risk factor for stroke. We examined the molecular mechanism of Ang II mediated cerebrovascular remodeling that involves the epidermal growth factor receptor (EGFR) pathway. Mutant EGFR mice (waved-2), their heterozygous control (wild-type [WT]), and C57BL/6J mice were infused with Ang II (1000 ng kg 1 min 1 ) or saline via osmotic minipumps for 28 days (n=8 per group). Eight of the Ang II infused C57BL/6J mice were cotreated with AG1478 (12 mg/kg per day, IP), a specific EGFR tyrosine kinase inhibitor. Systolic arterial pressure was measured by a tail-cuff method. Pressure and diameter of cerebral arterioles were measured through an open cranial window in anesthetized mice. Cross-sectional area of the wall was determined in pressurized fixed cerebral arterioles. Expression of phosphorylated EGFR (p-egfr), caveolin-1 (Cav-1), and c-src was determined by western blotting and immunohistochemistry. Mutation of EGFR or AG1478 treatment did not affect Ang II induced hypertension. Ang II increased the expression of p-egfr in WT mice, confirming the activation of EGFR. Ang II induced hypertrophy and inward remodeling of cerebral arterioles in WT mice. Hypertrophy, but not remodeling, was prevented in waved-2 and AG1478-treated C57BL/6J mice. Ang II increased p-egfr, Cav-1, and c-src expression in WT but not in waved-2 or AG1478-treated C57BL/6J mice. These results suggest that Ang II induced hypertrophy in cerebral arterioles involves EGFR-dependent signaling and may include Cav-1 and nonreceptor tyrosine kinase c-src. This signaling pathway seems to be limited to Ang II induced hypertrophy, but not inward remodeling, and is independent of blood pressure. (Hypertension. 2015;65: DOI: /HYPERTENSIONAHA ) Online Data Supplement Key Words: angiotensin II hypertrophy mice vascular remodeling Chronic hypertension is one of the most important modifiable risk factors for stroke and vascular dementia. 1,2 In the cerebral circulation, vascular remodeling in smaller resistance vessels during chronic hypertension, including hypertrophy and inward remodeling, can be both protective and detrimental. 3 Despite substantial efforts in the past few decades, the molecular mechanisms of vascular remodeling in the cerebral circulation are not completely understood. It is therefore of ultimate importance to study vascular remodeling in smaller resistance vessels to fully understand and provide effective treatment of stroke. The renin angiotensin system is activated in chronic hypertension. The system s main effector molecule, angiotensin II (Ang II), plays crucial roles in vascular smooth muscle cell (VSMC) growth and migration, which is critical in hypertrophy and inward remodeling in cerebral arterioles. These functions are mediated, in large part, by the G-protein coupled angiotensin type 1 receptor (AT 1 R). 4 Although AT 1 R lacks intrinsic tyrosine kinase activity, its activation nevertheless leads to tyrosine phosphorylation of multiple signaling proteins. As an example, AT 1 R has been shown to transactivate epidermal growth factor receptor (EGFR), a receptor tyrosine kinase that can be activated by various growth factors. 5 In addition, Ang II mediated proliferation and migration of SMC were found to depend on transactivation of EGFR. 6 Also of interest is that Ang II mediated EGFR transactivation in VSMC requires the presence of reactive oxygen species. 7 We have recently shown that reactive oxygen species generated by Nox2-containing nicotinamide adenine dinucleotide phosphate oxidase are critical in Ang II induced vascular remodeling in cerebral arterioles. 8 These findings strongly suggest that vascular remodeling in cerebral arterioles might involve Ang II and subsequent production of reactive oxygen species, leading to transactivation of EGFR. Therefore, the first goal in this study was to examine the role of EGFR signaling in Ang II mediated hypertrophy and inward remodeling in cerebral arterioles using both a genetic model of EGFR point mutation and a pharmacological inhibitor. Formation of caveolae may play a central role in transactivation of receptor tyrosine kinases by Ang II. Caveolae are plasma membrane microdomains (aka lipid rafts) where Received October 22, 2014; first decision November 3, 2014; revision accepted February 1, From the Department of Pathology, University of Iowa College of Medicine, Iowa City. Current address for S.-L.C.: Department of Neurological Sciences, University of Vermont, Burlington. The online-only Data Supplement is available with this article at /-/DC1. Correspondence to Gary L. Baumbach, Department of Pathology, University of Iowa Carver College of Medicine, 5231C RCP, 200 Hawkins Dr, Iowa City, IA g-baumbach@uiowa.edu 2015 American Heart Association, Inc. Hypertension is available at DOI: /HYPERTENSIONAHA

2 Chan et al EGFR on Ang II Hypertrophy in Cerebral Arterioles 807 EGFRs, as well as many other signaling molecules, such as c-src, are compartmentalized, in part, by forming complexes with Cav-1. 9 It has been proposed that the transactivation of EGFR is mediated by AT 1 R trafficking into caveolae. 10 In addition, c-src, the most abundant subtype of the Src family in the vasculature, has been shown to be essential in Ang II mediated transactivation of EGFR in VSMC. 11 This c-src dependent pathway also relies on the production of superoxide derived from nicotinamide adenine dinucleotide phosphate oxidase. 7 Thus, our second goal in this study was to determine whether Cav-1 and c-src are involved in EGFR-mediated, Ang II induced vascular remodeling in cerebral arterioles. Methods Materials and Methods are available in the online-only Data Supplement. Animals Breeding pairs of female heterozygous and male homozygous waved-2 mice were crossed per recommendation of Jackson Laboratory (Bar Harbor, ME) to generate animals for experimental use (Strain name: STOCK a/a Egfr wa2 /J; stock number: ). Waved-2 mice lose 90% of EGFR function. 12 These mice have curled hair and vibrissae at young age that disappear later in life and have abnormal aortic valve morphology. Heterozygous littermates (wild-type [WT]) were used as controls. All mice were genotyped before use. C57BL/6J mice were purchased from Jackson Laboratory. Animals were housed in pathogen-free facility at 24 C, exposed to 12 hours of light, and allowed free access of food and fluid. All procedures were approved by Institutional Animal Care and Use Committee of the University of Iowa and in agreement with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Treatments To study the effects of EGFR deficiency in a genetic model, WT and waved-2 mice were randomly divided into a treatment group that received Ang II (1000 ng kg 1 min 1 ; 28 days) via osmotic minipumps (model 1004; Durect, Cupertino, CA) and a control group that received saline (n=8 per group). In a second set of experiments, the effects of EGFR inhibition were studied using the pharmacological inhibitor AG1478 in C57BL/6J mice that were randomly divided into 2 treatment groups and a control group. The first treatment group (n=10) received Ang II (1000 ng kg 1 min 1 ) via osmotic minipumps for 28 days and AG1478 via intraperitoneal injections (12 mg/kg per day; Sigma, St Louis, MO). 13 This dosing regimen was expected to achieve plasma concentrations of AG1478 sufficient to significantly inhibit the activity of EGFR tyrosine kinase. 14 The second treatment group (n=10) received Ang II and injection of vehicle (DMSO diluted in saline). The control group (n=8) received saline via minipump only. Measurement of Cerebral Arteriolar Pressure and Diameter We measured pressure and diameter in first-order arterioles on the cerebrum in anesthetized mice through an open-skull preparation that we described previously in detail. 15 Determination of Cerebral Arteriolar Structure About 30 minutes after completion of surgery, baseline pressure and diameter of cerebral arterioles were measured. Arterioles were then suffused with artificial cerebrospinal fluid containing ethylenediaminetetraacetic acid (67 mmol/l), which produced maximal dilatation of cerebral arterioles. 16 Cross-sectional area of the arteriolar wall was determined histologically as described previously. 8,17 Statistical Analysis Differences between groups were determined by Student t test for 2 groups or 1-way ANOVA for 3 groups using Graph Pad Prism 6 (Graph Pad Software, San Diego, CA). Values were presented in mean±sem and were considered different when P<0.05. Results Ang II Activates EGFR in Cerebral Arteries Ang II has been shown to activate EGFR via phosphorylation in VSMC. 6 To determine whether Ang II activates EGFR in cerebral arteries, western blotting of p-egfr, the active form of EGFR, was performed. Ang II increased the density of p-egfr in cerebral arteries in WT mice (Figure 1A), suggesting Ang II increased phosphorylation of EGFR. Furthermore, immunohistochemical staining found that Ang II significantly increased the expression of p-egfr in cerebral arterioles in WT but not in waved-2 mice (Figure 1B). Isotype IgG control of p-egfr confirmed the antibody specificity (Figure S1 in the online-only Data Supplement). This result confirmed that Ang II activated EGFR in cerebral arterioles in WT mice and suggested that Ang II induced activation of EGFR expression was attenuated in waved-2 mice. Ang II Causes Hypertrophy of Cerebral Arterioles Through EGFR We have previously shown that Ang II is a determinant of hypertrophy in cerebral arterioles. 8 In this study, cross-sectional area of the cerebral arteriolar wall was significantly increased by Ang II in WT (537±41 versus 399±24 μm 2 in saline) but not in waved-2 mice (404±20 versus 418±27 μm 2 in saline; Figure 2). To confirm the role of EGFR, we used pharmacological inhibitor of EGFR tyrosine kinase, AG1478 in C57BL/6J mice. AG1478 abolished Ang II induced increase in cross-sectional area (356±19 versus 527±31 in Ang II alone; Figure 3A). Furthermore, AG1478 inhibited the Ang II induced increased expression of p-egfr in cerebral vasculature (Figure 3B). Additional isotype IgG control of p-egfr confirmed the antibody specificity in C57BL/6J mice (Figure S1). These results strongly demonstrate a critical role of EGFR in Ang II induced cerebral arteriolar hypertrophy. In addition to hypertrophy, Ang II has been shown to be a determinant of inward remodeling and increased distensibility in cerebral arterioles. 8,18 In this study, however, mutation or inhibition of EGFR neither affects baseline diameters (Table) nor attenuates the downward shift in the pressure diameter relationship (Figure S2) observed in maximally dilated cerebral arterioles of Ang II treated controls, suggesting that activation of EGFR did not contribute significantly to Ang II mediated inward remodeling. Stress strain relationship (Figure S3) was not affected by EGFR mutation. Although AG1478 had some effects to slightly left-shifted the curve, collectively wall distensibility was not significantly affected in cerebral arterioles. Molecular Mechanism of EGFR-Mediated, Ang II Induced Hypertrophy Cell culture studies have demonstrated that both Cav-1 and c-src are important signaling molecules in VSMC growth, 11,19 which is considered a mechanism for hypertrophy in cerebral arterioles. Protein expression of Cav-1 and c-src was significantly increased by Ang II in cerebral arteries from WT mice (Figure 4A). Moreover, although Ang II also increased the protein expression of Cav-1 and c-src in EGFR / mice, the increase was

3 808 Hypertension April 2015 Figure 1. Angiotensin II (Ang II) activates epidermal growth factor receptor (EGFR) in cerebral arterioles. Representative photograph and densitometry of phosphorylated EGFR (p-egfr) western blotting in cerebral arteries from wild-type (WT) mice with or without Ang II (A); and representative photographs and staining density of p-egfr immunohistochemistry in cerebral arterioles from WT and waved-2 mice treated with Ang II (B). Results are mean±sem of 6 to 8 mice. *P<0.05 vs WT. Scale bar, 10 μm. substantially smaller. Similarly, immunohistochemistry showed that strong staining density of Cav-1 and c-src in cerebral arterioles was only observed in Ang II treated WT but not in waved-2 mice (Figure 4B). Isotype IgG controls of Cav-1 and c-src confirmed the antibody specificity (Figure S1). These results support the concept that Cav-1 and c-src are involved in Ang II EGFR signaling in hypertrophy of cerebral arterioles. Deficiency of EGFR Does Not Affect Ang II Induced Hypertension Ang II may induce hypertrophy in cerebral arterioles through a direct or an indirect pressor effect. 18,20 Therefore, it is important to evaluate whether the effect of EGFR inhibition on hypertrophy is pressure dependent. We found that Ang II increased systemic blood pressure comparably between WT and waved-2 mice (Figure 5). Similarly, EGFR inhibitor Figure 2. Epidermal growth factor receptor mutation prevents cerebral arteriolar hypertrophy induced by angiotensin II (Ang II). Cross-sectional area of cerebral arteriolar wall in wild-type (WT) and waved-2 mice treated with saline or Ang II. Results are mean±sem of 8 mice. *P<0.05 vs saline. AG1478 did not have significant effect on Ang II induced hypertension. These results suggested that reduced hypertrophy in cerebral arterioles by EGFR deficiency was not because of a blood pressure lowering effect. Discussion Vascular remodeling is considered an adaptive response in chronic hypertension. Small cerebral arterioles undergo hypertrophy and inward remodeling to protect the vessel wall against increased wall stress. However, these structural changes also have negative effect. For example, smaller lumen diameter may negatively affect the vessel s ability to dilate and increase blood flow when needed. Because chronic hypertension is a major risk factor for stroke, understanding the molecular mechanisms of vascular remodeling induced by increased pressure can be useful for understanding the cause and better treatment of stroke. Therefore, we sought to determine the molecular mechanism of vascular remodeling in small cerebral arterioles, which has substantial vascular resistance and thought to be important for local blood flow control. We have several novel findings; first, we showed that phosphorylation of EGFR was increased in cerebral arterioles from mice treated with Ang II. Second, we demonstrated that EGFR activation was critical in Ang II induced hypertrophy in cerebral arterioles. EGFR activation, however, did not seem to involve in Ang II mediated inward remodeling and increased in wall distensibility. Third, Cav-1 and c-src, signaling molecules that heavily participated in Ang II induced hypertrophy in VSMC, may be involved in EGFR-dependent hypertrophy in cerebral arterioles. This pathway may be pressure-independent because EGFR mutation or AG1478 did not significantly affect Ang II induced hypertension.

4 Chan et al EGFR on Ang II Hypertrophy in Cerebral Arterioles 809 Figure 3. Pharmacological inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase prevents cerebral arteriolar hypertrophy induced by angiotensin II (Ang II). Cross-sectional area (A) and immunohistochemistry of phosphorylated EGFR (B) of cerebral arterioles from C57BL/6J mice treated with saline, Ang II or Ang II plus AG1478 (AG). Results are mean±sem of 6 to 8 mice. *P<0.05 vs saline; #P<0.05 vs Ang II alone. Scale bar, 10 μm. The renin angiotensin system is activated during chronic hypertension. Ang II, the major effector molecule of the renin angiotensin system, mediates hypertrophy and inward remodeling in cerebral arterioles, which is dependent on reactive oxygen species derived from Nox2-containing nicotinamide adenine dinucleotide phosphate oxidase. 8,15 We examined the role of EGFR in this study because previous studies in VSMC cultures showed that reactive oxygen species were critical in transactivation of EGFR. We demonstrated that Ang II increased the protein expression of p-egfr. Ang II induced hypertrophy in cerebral arterioles was inhibited by EGFR mutation or AG1478. These results strongly suggest, for the first time, that EGFR activation is essential in Ang II mediated cerebral arteriolar hypertrophy. In contrast, EGFR was not involved in Ang II mediated inward remodeling, which Table. supports the idea that molecular mechanisms of hypertrophy and inward remodeling are distinctive and independent. 15,21 The process through which Ang II transactivates EGFR in VSMC is known to involve multiple signaling events beginning with activation of AT 1 R. 22 Once activated, AT 1 R migrates into Cav-1 rich caveolae along with several other signaling molecules, including Cav-1, EGFR, c-src, and cabl. 19 This recruitment process seems to be reactive oxygen species dependent. 10 The end result is that Cav-1 acts as a scaffold protein that brings together the signaling events through which Ang II leads to EGFR transactivation. Based on these findings in VSMC, we sought to determine whether Cav-1 and c-src are also involved in Ang II induced transactivation of EGFR and hypertrophy in cerebral arterioles. We found that Ang II increased the expression of Cav-1 and c-src in WT but not in Cerebral Arteriolar Pressures and Diameters (at 40 mm Hg); Arterial Blood Gases During Experiments in Anesthetized Mice Parameters WT Saline WT Ang II Waved-2 Saline Waved-2 Ang II C57BL/6J Saline C57BL/6J Ang II C57BL/6J Ang II+AG Cerebral arteriolar pressure Systolic, mm Hg 36±5 34±4 35±4 32±3 36±5 53±5 51±3 Diastolic, mm Hg 24±3 24±2 23±4 26±3 24±3 39±5 38±3 Mean, mm Hg 30±4 29±3 29±4 29±3 30±4 44±5 42±3 Pulse, mm Hg 12±3 10±2 11±1 9±2 12±3 14±2 14±1 Diameter, μm 69±3 58±3* 68±4 59±3* 70±4 54±3* 56±3* Arterial blood gas ph 7.35± ± ± ± ± ± ±0.01 PCO 2 28±4 30±2 22±2 24±3 28±4 32±4 26±2 PO 2 117±5 104±4 119±8 107±9 117±5 111±10 119±7 Age, wk 16.0± ± ± ± ± ± ±0.3 Weight, g 24.1± ± ± ± ± ± ±0.8 n Values are mean±sem. AG: AG1478. P<0.05 versus corresponding saline controls. Ang II indicates angiotensin II; and WT, wild-type. *P<0.05 vs saline.

5 810 Hypertension April 2015 Figure 4. Caveolin-1 (Cav-1) and c-src are involved in angiotensin II (Ang II) epidermal growth factor receptor (EGFR) signaling. Representative photographs and densitometry of Cav-1 and c-src western blotting in cerebral arteries from wild-type (WT) and waved-2 mice with or without Ang II (A); and representative photographs and straining density of Cav-1 and c-src immunohistochemistry in cerebral arterioles from WT and waved-2 mice with or without Ang II (B). Results are mean±sem of 6 to 8 mice. *P<0.05 vs control; #P<0.05 vs waved-2 with Ang II. Scale bar, 10 μm. waved-2 mice, which suggests that these signaling molecules indeed may play a role in Ang II induced cerebral arteriolar hypertrophy. Although how Cav-1 is involved in this signaling pathway is not known, the result of this study may suggest that Cav-1 has pro-proliferation properties in cerebral arterioles. Although Ang II may induce cerebral vascular hypertrophy through an AT 1 R-dependent mechanism independently of its pressor effect, one must consider the possibility that a pressordependent mechanism also contributed to our finding in this study of Ang II. 20 In this study, we found that EGFR mutation or AG1478 treatment did not blunt the pressor effect of Ang II, but nevertheless prevented Ang II mediated hypertrophy. Previous studies showed that AG1478 either unchanged or decreased the blood pressure in anesthetized rats. 23,24 Because inhibition of EGFR did not affect blood pressure in this study, it is more likely that Ang II mediates hypertrophy through a direct AT 1 R-EGFR dependent pathway, rather than other indirect mechanisms. It is noteworthy that although genetic mutation or AG1478 did not affect hypertension induced by 4-week treatment of Ang II in this study, previous studies showed that other means of EGFR inhibition reduced systemic blood pressure in different animal models, suggesting that EGFR may be involved in blood pressure regulation under particular conditions. For Figure 5. Epidermal growth factor receptor mutation or inhibition does not affect angiotensin II (Ang II) induced hypertension. Conscious systolic blood pressures measured by tail-cuff method in wild-type (WT), waved-2, and C57BL/6J mice treated with saline, Ang II or Ang II plus AG1478 (AG). Results are mean±sem of 6 to 8 mice. *P<0.05 vs saline.

6 Chan et al EGFR on Ang II Hypertrophy in Cerebral Arterioles 811 example, hypertension induced by a lower dose of Ang II for 2 weeks was reduced by 15 mm Hg by EGFR antisense in rats. 25 Another study showed that a different pharmacological EGFR inhibitor, PKI-166, reduced the blood pressure in nephrectomized rats after 8 weeks. 26 A few limitations in this study are noteworthy. First, we were not able to detect the pressor effect of Ang II in cerebral arterioles (Table) although an increase was observed in systemic pressure. The likely explanation for this apparent discrepancy is that systemic pressure was measured in unanesthetized mice using the tail-cuff method, whereas cerebral arteriolar pressure was measured in anesthetized mice using a servo-null system. Anesthesia is known to lower blood arterial pressure in mice and blunt responses to pressor agents. 27 Despite this, the evaluation of long-term structural changes in cerebral arterioles by Ang II should not be affected under this condition. Second, the use of western blotting to determine protein expression of p-egfr, Cav-1, and c-src was limited to larger cerebral arteries because cerebral arterioles contain insufficient amounts of protein. However, we have further confirmed the expression and location of these proteins in cerebral arterioles using immunohistochemistry. Third, although Cav-1 and c-src may be important signaling molecules, further studies are needed to determine exactly how these proteins interact within the signaling pathway of Ang II EGFR dependent hypertrophy. Perspectives Ang II is a major mediator of vascular structural changes in cerebral arterioles during chronic hypertension, which is one of the most important risk factors for stroke. The molecular mechanism of this process, however, is not completely understood. EGFR-dependent signaling is likely involved in Ang II mediated cerebral arteriolar hypertrophy but not inward remodeling. Cav-1 and c-src may also be involved and deserved more in-depth investigations. Acknowledgments We thank Tom Gerhold for excellent technical assistance on open cranial window experiments and genotyping of genetic mice. Sources of Funding This work was supported by Department of Pathology, The University of Iowa, National Institutes of Health Grant NS72628 and HL62984, and American Heart Association (Midwest Affiliate) Grant-in-Aid Award 09GRNT Dr Chan was a recipient of American Heart Association Post-doctoral Fellowship Z. None. Disclosures References 1. Sacco RL, Wolf PA, Gorelick PB. Risk factors and their management for stroke prevention: outlook for 1999 and beyond. Neurology. 1999;53(7 suppl 4):S15 S Verdelho A, Madureira S, Ferro JM, et al; LADIS Study. Differential impact of cerebral white matter changes, diabetes, hypertension and stroke on cognitive performance among non-disabled elderly. The LADIS study. J Neurol Neurosurg Psychiatry. 2007;78: doi: / jnnp Izzard AS, Rizzoni D, Agabiti-Rosei E, Heagerty AM. Small artery structure and hypertension: adaptive changes and target organ damage. J Hypertens. 2005;23: Higuchi S, Ohtsu H, Suzuki H, Shirai H, Frank GD, Eguchi S. Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology. Clin Sci (Lond). 2007;112: doi: /CS Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T. Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem. 1998;273: Yang X, Zhu MJ, Sreejayan N, Ren J, Du M. Angiotensin II promotes smooth muscle cell proliferation and migration through release of heparin-binding epidermal growth factor and activation of EGF-receptor pathway. Mol Cells. 2005;20: Ushio-Fukai M, Griendling KK, Becker PL, Hilenski L, Halleran S, Alexander RW. Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2001;21: Chan SL, Baumbach GL. Deficiency of Nox2 prevents angiotensin II-induced inward remodeling in cerebral arterioles. Front Physiol. 2013;4:133. doi: /fphys Krajewska WM, Masłowska I. Caveolins: structure and function in signal transduction. Cell Mol Biol Lett. 2004;9: Zuo L, Ushio-Fukai M, Ikeda S, Hilenski L, Patrushev N, Alexander RW. Caveolin-1 is essential for activation of Rac1 and NAD(P)H oxidase after angiotensin II type 1 receptor stimulation in vascular smooth muscle cells: role in redox signaling and vascular hypertrophy. Arterioscler Thromb Vasc Biol. 2005;25: doi: /01. ATV Bokemeyer D, Schmitz U, Kramer HJ. Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor. Kidney Int. 2000;58: doi: /j t x. 12. Luetteke NC, Phillips HK, Qiu TH, Copeland NG, Earp HS, Jenkins NA, Lee DC. The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase. Genes Dev. 1994;8: Ishii Y, Fujimoto S, Fukuda T. Gefitinib prevents bleomycin-induced lung fibrosis in mice. Am J Respir Crit Care Med. 2006;174: doi: /rccm OC. 14. Ellis AG, Doherty MM, Walker F, Weinstock J, Nerrie M, Vitali A, Murphy R, Johns TG, Scott AM, Levitzki A, McLachlan G, Webster LK, Burgess AW, Nice EC. Preclinical analysis of the analinoquinazoline AG1478, a specific small molecule inhibitor of EGF receptor tyrosine kinase. Biochem Pharmacol. 2006;71: doi: /j.bcp Baumbach GL, Sigmund CD, Faraci FM. Cerebral arteriolar structure in mice overexpressing human renin and angiotensinogen. Hypertension. 2003;41: Baumbach GL, Dobrin PB, Hart MN, Heistad DD. Mechanics of cerebral arterioles in hypertensive rats. Circ Res. 1988;62: Baumbach GL, Siems JE, Heistad DD. Effects of local reduction in pressure on distensibility and composition of cerebral arterioles. Circ Res. 1991;68: Chillon JM, Baumbach GL. Effects of an angiotensin-converting enzyme inhibitor and a beta-blocker on cerebral arterioles in rats. Hypertension. 1999;33: Ushio-Fukai M, Zuo L, Ikeda S, Tojo T, Patrushev NA, Alexander RW. cabl tyrosine kinase mediates reactive oxygen species- and caveolindependent AT1 receptor signaling in vascular smooth muscle: role in vascular hypertrophy. Circ Res. 2005;97: doi: /01. RES F Griffin SA, Brown WC, MacPherson F, McGrath JC, Wilson VG, Korsgaard N, Mulvany MJ, Lever AF. Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism. Hypertension. 1991;17: Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic hypertension. Hypertension. 1989;13(6 Pt 2): Eguchi S, Inagami T. Signal transduction of angiotensin II type 1 receptor through receptor tyrosine kinase. Regul Pept. 2000;91: Escano CS Jr, Keever LB, Gutweiler AA, Andresen BT. Angiotensin II activates extracellular signal-regulated kinase independently of receptor tyrosine kinases in renal smooth muscle cells: implications for blood pressure regulation. J Pharmacol Exp Ther. 2008;324: doi: / jpet

7 812 Hypertension April Beaucage P, Moreau P. EGF receptor transactivation in angiotensin II and endothelin control of vascular protein synthesis in vivo. J Cardiovasc Pharmacol. 2004;44(suppl 1):S20 S Kagiyama S, Eguchi S, Frank GD, Inagami T, Zhang YC, Phillips MI. Angiotensin II-induced cardiac hypertrophy and hypertension are attenuated by epidermal growth factor receptor antisense. Circulation. 2002;106: Ulu N, Mulder GM, Vavrinec P, Landheer SW, Duman-Dalkilic B, Gurdal H, Goris M, Duin M, van Dokkum RP, Buikema H, van Goor H, Henning RH. Epidermal growth factor receptor inhibitor PKI-166 governs cardiovascular protection without beneficial effects on the kidney in hypertensive 5/6 nephrectomized rats. J Pharmacol Exp Ther. 2013;345: doi: /jpet Baumbach GL, Sigmund CD, Faraci FM. Structure of cerebral arterioles in mice deficient in expression of the gene for endothelial nitric oxide synthase. Circ Res. 2004;95: doi: /01. RES Novelty and Significance What Is New? Epidermal growth factor receptor (EGFR) dependent signaling is essential in cerebral arteriolar hypertrophy, not in inward remodeling, induced by angiotensin II. The signaling cascade may also involve Cav-1 and c-src. What Is Relevant? Angiotensin II induced cerebral arteriolar hypertrophy and inward remodeling are not completely understood and thought to be distinctive and independent. This study unmasks an important mechanism involving EGFR signaling that is only responsible for hypertrophy. Summary Angiotensin II induces hypertrophy in cerebral arterioles through EGFR activation and EGFR-dependent signaling pathway, possibly also involves Cav-1 and c-src. EGFR activation seems to be only responsible for angiotensin II mediated hypertrophy but not for inward remodeling and has no effect on blood pressure.