Advertisement

Stretch-induced Retinal Vascular Endothelial Growth Factor Expression Is Mediated by Phosphatidylinositol 3-Kinase and Protein Kinase C (PKC)-ζ but Not by Stretch-induced ERK1/2, Akt, Ras, or Classical/Novel PKC Pathways*

Open AccessPublished:November 01, 2001DOI:https://doi.org/10.1074/jbc.M105336200
      Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC)-ζ but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt, or Ras despite their activation by stretch. Cardiac profile cyclic stretch at 60 cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-ζ activity. Signaling pathways were explored using inhibitors of PKC, MEK1/2, and PI 3-kinase; adenovirus-mediated overexpression of ERK, PKC-α, PKC-δ, PKC-ζ, and Akt; and dominant negative (DN) mutants of ERK, PKC-ζ, Ras, PI 3-kinase and Akt. Although stretch activated ERK1/2 through a Ras- and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of DN ERK and Ras had no effect on VEGF expression in these cells. In contrast, DN PI 3-kinase as well as pharmacologic inhibitors of PI 3-kinase blocked stretch-induced VEGF expression. Although stretch-induced PI 3-kinase activation increased both Akt phosphorylation and activity of PKC-ζ, VEGF expression was dependent on PKC-ζ but not Akt. In addition, PKC-ζ did not mediate stretch-induced ERK1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI 3-kinase-mediated activation of PKC-ζ that is independent of stretch-induced activation of ERK1/2, classical/novel PKC isoforms, Ras, or Akt. This mechanism may play a role in the well documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.
      VEGF
      vascular endothelial growth factor
      PKC
      protein kinase C
      PI
      phosphatidylinositol
      ERK
      extracellular signal-regulated kinase
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      KDR or VEGF-R2
      tyrosine kinase insert domain-containing VEGF receptor
      BRPC
      bovine retinal pericyte
      ca
      constitutively active
      mt
      mutant
      DN
      dominant negative
      PDGF
      platelet-derived growth factor
      PDGFR-B
      PDGF receptor B
      GFP
      green fluorescent protein
      One in four American adults has hypertension, while 5.9% of the United States population (over 15 million people) have diabetes. Diabetic retinopathy is the leading cause of new onset blindness in the United States among working age individuals (
      • National Society to Prevent Blindness
      Data Analysis. Vision Problems in the US: Facts and Figures..
      ) and is exacerbated by coexistent systemic hypertension (
      • Klein R.
      • Klein B.E.
      • Moss S.E.
      • Cruickshanks K.J.
      ,
      • Wan N.W.
      • Letchuman R.
      • Noraini N.
      • Ropilah A.R.
      • Zainal M.
      • Ismail I.S.
      • Wan M.W.
      • Faridah I.
      • Singaraveloo M.
      • Sheriff I.H.
      • Khalid B.A.
      ,
      • Agardh C.D.
      • Agardh E.
      • Torffvit O.
      ). Sight-threatening diabetic retinopathy is characterized by development of retinal neovascularization and/or retinal vascular permeability (
      • Aiello L.P.
      • Gardner T.W.
      • King G.L.
      • Blankenship G.W.
      • Cavallerano J.
      • Ferris F.
      • Klein R.
      ). Hypertension increases the risk of retinopathy progression, development of neovascularization (
      • Klein R.
      • Klein B.E.
      • Moss S.E.
      • Cruickshanks K.J.
      ,
      • Rosenn B.
      • Miodovnik M.
      • Kranias G.
      • Khoury J.
      • Combs C.A.
      • Mimouni F.
      • Siddiqi T.A.
      • Lipman M.J.
      ,
      • Roy M.S.
      ), and retinal vascular permeability (
      • El-Asrar A.M.
      • Al-Rubeaan K.A.
      • Al-Amro S.A.
      • Kangave D.
      • Moharram O.A.
      ,
      • Lopes de Faria J.M.
      • Jalkh A.E.
      • Trempe C.L.
      • McMeel J.W.
      ) by up to 3-fold. Blood pressure control reduces both retinopathy progression and severe visual loss (
      UK Prospective Diabetes Study Group
      ). Even in normotensive diabetic patients retinopathy is associated with higher systolic blood pressure (
      • Le Floch J.P.
      • Christin S.
      • Bertherat J.
      • Perlemuter L.
      • Hazard J.
      ). Other vision-threatening conditions such as hypertensive retinopathy (
      • Tso M.O.
      • Jampol L.M.
      ) and age-related macular degeneration are also aggravated by hypertension (
      Macular Photocoagulation Study Group
      ).
      Although the mechanisms underlying the exacerbation of these conditions by hypertension are not fully understood, vascular endothelial growth factor (VEGF)1 has been strongly implicated as a primary mediator of ocular complications in diabetes and age-related macular degeneration. VEGF is a hypoxia-induced, endothelial cell-selective mitogen (
      • Shweiki D.
      • Itin A.
      • Soffer D.
      • Keshet E.
      ,
      • Shweiki D.
      • Itin A.
      • Neufeld G.
      • Gitay-Goren H.
      • Keshet E.
      ,
      • Shweiki D.
      • Neeman M.
      • Itin A.
      • Keshet E.
      ) also called vascular permeability factor after its potent ability to induce vasopermeability (
      • Senger D.R.
      • Connolly D.T.
      • Van de Water L.
      • Feder J.
      • Dvorak H.F.
      ). VEGF is the principal stimuli for intraocular neovascularization and retinal vascular permeability in diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration, and numerous other conditions (
      • Aiello L.P.
      • Avery R.L.
      • Arrigg P.G.
      • Keyt B.A.
      • Jampel H.D.
      • Shah S.T.
      • Pasquale L.R.
      • Thieme H.
      • Iwamoto M.A.
      • Park J.E.
      ,
      • Aiello L.P.
      • Northrup J.M.
      • Keyt B.A.
      • Takagi H.
      • Iwamoto M.A.
      ,
      • Pierce E.A.
      • Avery R.L.
      • Foley E.D.
      • Aiello L.P.
      • Smith L.E.
      ,
      • Simorre-Pinatel V.
      • Guerrin M.
      • Chollet P.
      • Penary M.
      • Clamens S.
      • Malecaze F.
      • Plouet J.
      ,
      • Adamis A.P.
      • Shima D.T.
      • Yeo K.T.
      • Yeo T.K.
      • Brown L.F.
      • Berse B.
      • D'Amore P.A.
      • Folkman J.
      ,
      • Frank R.N.
      ,
      • Amin R.H.
      • Frank R.N.
      • Kennedy A.
      • Eliott D.
      • Puklin J.E.
      • Abrams G.W.
      ,
      • Ishibashi T.
      • Hata Y.
      • Yoshikawa H.
      • Nakagawa K.
      • Sueishi K.
      • Inomata H.
      ,
      • Kvanta A.
      • Algvere P.V.
      • Berglin L.
      • Seregard S.
      ,
      • Pe'er J.
      • Folberg R.
      • Itin A.
      • Gnessin H.
      • Hemo I.
      • Keshet E.
      ). VEGF exerts its action through the high affinity tyrosine kinase insert domain-containing receptor (KDR, VEGF-R2) (
      • Fong G.H.
      • Rossant J.
      • Gertsenstein M.
      • Breitman M.L.
      ,
      • Shalaby F.
      • Rossant J.
      • Yamaguchi T.P.
      • Gertsenstein M.
      • Wu X.F.
      • Breitman M.L.
      • Schuh A.C.
      ).In vivo, hypertension can increase large artery (
      • Safar M.E.
      • Peronneau P.A.
      • Levenson J.A.
      • Toto-Moukouo J.A.
      • Simon A.C.
      ) and retinal artery distention (
      • Houben A.J.
      • Canoy M.C.
      • Paling H.A.
      • Derhaag P.J.
      • de Leeuw P.W.
      ) as much as 15 and 35%, respectively. Mechanical stretch induces VEGF expression in rat ventricular myocardium (
      • Li J.
      • Hampton T.
      • Morgan J.P.
      • Simons M.
      ), rat cardiac myocytes (
      • Seko Y.
      • Takahashi N.
      • Shibuya M.
      • Yazaki Y.
      ), human mesangial cells (
      • Gruden G.
      • Thomas S.
      • Burt D.
      • Lane S.
      • Chusney G.
      • Sacks S.
      • Viberti G.
      ), and cultured retinal pigment epithelial cells (
      • Seko Y.
      • Fujikura H.
      • Pang J.
      • Tokoro T.
      • Shimokawa H.
      ). Recently we reported that mechanical stretch induced expression of VEGF and its receptors in retinal endothelial cells (
      • Suzuma I.
      • Hata Y.
      • Clermont A.
      • Pokras F.
      • Rook S.L.
      • Suzuma K.
      • Feener E.P.
      • Aiello L.P.
      ) and demonstrated that retinal expression of VEGF and VEGF-R2 was increased during hypertension in vivo.
      The molecular mechanisms underlying stretch-induced VEGF expression have not been studied extensively. Stretch rapidly activates a plethora of second messenger pathways including tyrosine kinases, p21ras, extracellular signal-regulated kinase (ERK), S6 kinase, protein kinase C (PKC), phospholipases C and D, and the P450 pathway (
      • Ishida T.
      • Takahashi M.
      • Corson M.A.
      • Berk B.C.
      ,
      • Sadoshima J.
      • Izumo S.
      ). Mechanical stretch can also regulate protein synthesis and the activity of numerous factors including NO (
      • Ziegler T.
      • Silacci P.
      • Harrison V.J.
      • Hayoz D.
      ), endothelin-1 (
      • Yamazaki T.
      • Komuro I.
      • Kudoh S.
      • Zou Y.
      • Shiojima I.
      • Hiroi Y.
      • Mizuno T.
      • Maemura K.
      • Kurihara H.
      • Aikawa R.
      • Takano H.
      • Yazaki Y.
      ), platelet-derived growth factor (
      • Hu Y.
      • Bock G.
      • Wick G.
      • Xu Q.
      ), fibroblast growth factor (
      • Park J.M.
      • Borer J.G.
      • Freeman M.R.
      • Peters C.A.
      ,
      • Marrero M.B.
      • Schieffer B.
      • Paxton W.G.
      • Heerdt L.
      • Berk B.C.
      • Delafontaine P.
      • Bernstein K.E.
      ), and angiotensin II (
      • Tamura K.
      • Umemura S.
      • Nyui N.
      • Hibi K.
      • Ishigami T.
      • Kihara M.
      • Toya Y.
      • Ishii M.
      ). Cyclic stretch can increase nerve growth factor in cultured urinary tract smooth muscle cells, an effect blocked by prolonged exposure to phorbol ester resulting in down-regulation of multiple PKC isoforms including α, β, δ, ε, and ζ (
      • Persson K.
      • Sando J.J.
      • Tuttle J.B.
      • Steers W.D.
      ).
      Of the numerous isoforms of PKC involved in the diverse signaling pathways of diabetes complications (
      • Koya D.
      • King G.L.
      ,
      • Bursell S.E.
      • Takagi C.
      • Clermont A.C.
      • Takagi H.
      • Mori F.
      • Ishii H.
      • King G.L.
      ,
      • King G.L.
      • Ishii H.
      • Koya D.
      ) and tumor angiogenesis (
      • Pal S.
      • Claffey K.P.
      • Cohen H.T.
      • Mukhopadhyay D.
      ,
      • Pal S.
      • Claffey K.P.
      • Dvorak H.F.
      • Mukhopadhyay D.
      ) PKC-ζ has been implicated in the regulation of VEGF expression (
      • Pal S.
      • Claffey K.P.
      • Cohen H.T.
      • Mukhopadhyay D.
      ,
      • Pal S.
      • Claffey K.P.
      • Dvorak H.F.
      • Mukhopadhyay D.
      ). PKC-ζ is an atypical isoform lacking the Ca2+binding C2 domain and with only one cysteine-rich zinc finger-like motif in the diacylglycerol binding C1 domain (
      • Ono Y.
      • Fujii T.
      • Ogita K.
      • Kikkawa U.
      • Igarashi K.
      • Nishizuka Y.
      ). Thus, PKC-ζ does not bind Ca2+ and is not activated by diacylglycerol or phorbol esters (
      • Nakanishi H.
      • Exton J.H.
      ). PKC-ζ is activated by several lipid mediators including phosphatidic acid (
      • Nakanishi H.
      • Exton J.H.
      ) and phosphatidylinositol 3,4,5-trisphosphate (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ). Nevertheless, PKC-ζ activity is important in mitogenesis, protein synthesis, cell survival, and regulation of transcription (
      • Berra E.
      • Diaz-Meco M.T.
      • Dominguez I.
      • Municio M.M.
      • Sanz L.
      • Lozano J.
      • Chapkin R.S.
      • Moscat J.
      ,
      • Chou M.M.
      • Hou W.
      • Johnson J.
      • Graham L.K.
      • Lee M.H.
      • Chen C.S.
      • Newton A.C.
      • Schaffhausen B.S.
      • Toker A.
      ).
      Expression of VEGF in response to Ras (
      • Pal S.
      • Datta K.
      • Khosravi-Far R.
      • Mukhopadhyay D.
      ), von Hippel-Lindau tumor suppressor gene (
      • Pal S.
      • Claffey K.P.
      • Dvorak H.F.
      • Mukhopadhyay D.
      ,
      • Shih S.C.
      • Mullen A.
      • Abrams K.
      • Mukhopadhyay D.
      • Claffey K.P.
      ), and transcription factor SP1 (
      • Pal S.
      • Claffey K.P.
      • Cohen H.T.
      • Mukhopadhyay D.
      ) is dependent upon PKC-ζ and subsequent ERK1/2 activation. Ras-induced VEGF expression in human fibrosarcoma and renal cell carcinoma cell lines is almost totally dependent on PKC-ζ activity, which is mediated through both Raf-dependent and Raf-independent pathways (
      • Pal S.
      • Datta K.
      • Khosravi-Far R.
      • Mukhopadhyay D.
      ). PKC-ζ has also been reported to mediate the downstream proliferative effect of VEGF (
      • Wellner M.
      • Maasch C.
      • Kupprion C.
      • Lindschau C.
      • Luft F.C.
      • Haller H.
      ).
      In this study, we examined the molecular mechanism of stretch-induced VEGF expression in retinal cells. These data are the first to demonstrate that stretch-induced VEGF expression is mediated by phosphatidylinositol (PI) 3-kinase and PKC-ζ in a manner independent of ERK1/2, Akt, or Ras. Thus, stretch-induced VEGF expression may be distinct from other pathways mediating VEGF expression, and theoretically, PI 3-kinase and PKC-ζ inhibitors may have therapeutic benefit in ameliorating the well documented exacerbation of ocular diseases by concomitant hypertension.

      DISCUSSION

      Our data demonstrate that cyclic stretch in retinal microvascular pericytes activates PI 3-kinase, classical/novel and atypical isoforms of PKC, ERK1/2, and Akt. In addition, stretch-induced ERK1/2 activation is predominantly Ras-dependent but PKC-ζ-independent. In contrast, stretch-induced VEGF expression is dependent on PI 3-kinase and PKC-ζ but independent of ERK1/2, classical/novel PKC isoforms, and Ras activity (Fig. 12).
      Figure thumbnail gr12
      Figure 12Mechanism of stretch-induced VEGF mRNA expression in bovine retinal pericytes. Schematic representation of potential mechanism for stretch-induced VEGF mRNA expression as supported by the data presented. Despite stretch-induced activation of all pathways listed, stretch-induced VEGF expression in retinal capillary pericytes is not primarily mediated by ERK1/2, Akt, or Ras but rather involves PI 3-kinase-mediated activation of PKC-ζ.
      The time course of VEGF expression in response to static and cyclic stretch in retinal pericytes was similar to that observed in retinal endothelial cells, although the magnitude of the response was approximately one-third of that in endothelial cells (
      • Suzuma I.
      • Hata Y.
      • Clermont A.
      • Pokras F.
      • Rook S.L.
      • Suzuma K.
      • Feener E.P.
      • Aiello L.P.
      ). Cyclic stretch induced rapid increases in ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-ζ activity. However, the ERK1/2 independence of stretch-induced VEGF expression was substantiated by several findings. Stretch-induced VEGF mRNA expression was not suppressed by either PD98059 or adenovirus infection with dominant negative ERK. Overexpression of wild type ERK did not increase basal or stretch-induced VEGF expression. Furthermore, stretch-induced ERK1/2 activation was mediated by classical/novel isoforms of PKC and Ras (as evidenced by inhibition of the response by classical/novel PKC isoforms inhibitor GF109203X and overexpression of dominant negative Ras) but not mediated by PI 3-kinase, tyrosine kinases, or PKC-ζ (as evidenced by lack of response to wortmannin and LY294002, lack of response to genistein, or overexpression of wild type and dominant negative PKC-ζ, respectively). In contrast, the opposite results were obtained when evaluating these interventions on stretch-induced VEGF expression. These data demonstrate that, although stretch activates several signaling pathways, VEGF expression is mediated by PI 3-kinase and PKC-ζ in an ERK-, Ras- and classical/novel PKC isoform-independent manner. In addition, direct modulation of ERK may not be adequate in itself to alter VEGF expression in these cells as evidenced by the lack of effect of ERK1/2 inhibitors and wild type or dominant negative ERK expression. It should be noted, however, that overexpression of wild type ERK1/2 might not have a major impact on the basal state if ERK is not significantly activated.
      The ERK independence of stretch-induced or basal VEGF expression is surprising. ERK has been reported as important in VEGF expression induced by starvation in human colon carcinoma cells (
      • Jung Y.D.
      • Nakano K.
      • Liu W.
      • Gallick G.E.
      • Ellis L.M.
      ), v-ras, v-raf, and c-myctransformation of rat liver epithelial cells (
      • Okajima E.
      • Thorgeirsson U.P.
      ), phorbol 12-myristate 13-acetate treatment in human glioblastoma U373 cells (
      • Shih S.C.
      • Mullen A.
      • Abrams K.
      • Mukhopadhyay D.
      • Claffey K.P.
      ), Ras expression in human fibrosarcoma and renal cell carcinoma cell lines (
      • Pal S.
      • Datta K.
      • Khosravi-Far R.
      • Mukhopadhyay D.
      ), endothelin stimulation of human vascular smooth muscle cells (
      • Pedram A.
      • Razandi M.
      • Hu R.M.
      • Levin E.R.
      ), and von Hippel-Lindau tumor suppressor gene action (
      • Pal S.
      • Claffey K.P.
      • Dvorak H.F.
      • Mukhopadhyay D.
      ). Hypoxic induction of VEGF may also involve ERK since inhibition of Raf-1 markedly reduces VEGF induction (
      • Mukhopadhyay D.
      • Tsiokas L.
      • Zhou X.M.
      • Foster D.
      • Brugge J.S.
      • Sukhatme V.P.
      ); however, hypoxia can be additive to VEGF expression induced by ERK1/2 activation in hamster fibroblasts where a single inhibitor of ERK did not suppress hypoxia-induced VEGF expression (
      • Milanini J.
      • Vinals F.
      • Pouyssegur J.
      • Pages G.
      ). The ERK independence observed in our system suggests that VEGF expression in response to different stimuli may be mediated by a variety of signaling pathways and/or may reflect a potential uniqueness of retinal pericytes.
      To our knowledge, the activation of PKC-ζ by stretch has not been previously documented. The importance of the atypical PKC-ζ isoform in mediating stretch-induced VEGF expression was underscored by several findings. PKC-ζ protein expression was present in retinal endothelial cells and present in even higher amounts in retinal pericytes. PKC-ζ activity was increased nearly 3-fold by cyclic stretch. Stretch-induced VEGF expression was inhibited by expression of dominant negative PKC-ζ and increased by overexpression of wild type PKC-ζ. In contrast, overexpression of wild type classical PKC-α isoform or novel PKC-δ isoform did not effect VEGF expression. The activation of PKC-ζ within 15 min of stretch onset is consistent with previous time course data for PKC-ζ activation following exposure to insulin (10–20 min) (
      • Standaert M.L.
      • Galloway L.
      • Karnam P.
      • Bandyopadhyay G.
      • Moscat J.
      • Farese R.V.
      ), nerve growth factor (9–15 min) (
      • Neri L.M.
      • Martelli A.M.
      • Borgatti P.
      • Colamussi M.L.
      • Marchisio M.
      • Capitani S.
      ), or hypoxia-reperfusion (15 min) (
      • Mizukami Y.
      • Kobayashi S.
      • Uberall F.
      • Hellbert K.
      • Kobayashi N.
      • Yoshida K.
      ).
      In other systems, including insulin-stimulated rat adipocytes (
      • Sajan M.P.
      • Standaert M.L.
      • Bandyopadhyay G.
      • Quon M.J.
      • Burke Jr., T.R.
      • Farese R.V.
      ), reoxygenation of rat cardiomyocytes (
      • Mizukami Y.
      • Kobayashi S.
      • Uberall F.
      • Hellbert K.
      • Kobayashi N.
      • Yoshida K.
      ), and endotoxin-treated human alveolar macrophages (
      • Monick M.M.
      • Carter A.B.
      • Flaherty D.M.
      • Peterson M.W.
      • Hunninghake G.W.
      ), PI 3-kinase activation induces ERK activity through a PKC-ζ-mediated pathway. However, our data suggest that stretch-induced activation of ERK1/2 in retinal pericytes is mediated by a different mechanism since inhibition of PKC-ζ using dominant negative adenovirus did not prevent stretch-induced ERK1/2 phosphorylation.
      Although these are the first studies to evaluate the role of PKC-ζ in stretch-induced VEGF expression, PKC-ζ has been previously implicated as a modulator of VEGF (
      • Pal S.
      • Claffey K.P.
      • Cohen H.T.
      • Mukhopadhyay D.
      ,
      • Pal S.
      • Claffey K.P.
      • Dvorak H.F.
      • Mukhopadhyay D.
      ). Overexpression of PKC-ζ in human glioblastoma U373 cells increased VEGF mRNA expression (
      • Shih S.C.
      • Mullen A.
      • Abrams K.
      • Mukhopadhyay D.
      • Claffey K.P.
      ). The von Hippel-Lindau tumor suppressor gene has been shown to form cytoplasmic complexes with PKC-δ and PKC-ζ, preventing their translocation to the cell membrane and reducing the constitutive overexpression of VEGF characteristically observed in sporadic renal cell carcinomas (
      • Pal S.
      • Claffey K.P.
      • Dvorak H.F.
      • Mukhopadhyay D.
      ). In addition, PKC-ζ binds and phosphorylates transcription factor SP1 in renal cell carcinomas, resulting in VEGF expression. Ras-induced VEGF expression in human fibrosarcoma and renal cell carcinoma cell lines is almost totally dependent on PKC-ζ activity (
      • Pal S.
      • Datta K.
      • Khosravi-Far R.
      • Mukhopadhyay D.
      ). However, as discussed above, ERK was an important component of these pathways.
      The role of PI 3-kinase in stretch-induced VEGF expression and Akt phosphorylation was supported by the inhibitory effect of two different PI 3-kinase inhibitors (wortmannin and LY294002) and dominant negative expression of the p85 subunit of PI 3-kinase. In addition, wortmannin completely inhibited stretch-induced PKC-ζ activity. However, Akt did not appear to mediate stretch-induced VEGF expression as expression of dominant negative or constitutively active Akt had no effect. This finding differs from that observed in chicken cells where overexpression of myristylated Akt increased basal VEGF expression and restored VEGF expression in cells after PI 3-kinase inhibition (
      • Jiang B.H.
      • Zheng J.Z.
      • Aoki M.
      • Vogt P.K.
      ). Thus, the role of Akt in mediating VEGF expression may be cell type- and/or stimuli-dependent. Our studies do not eliminate the possibility that stretch-induced Akt may be involved in late stages of VEGF expression (
      • Laughner E.
      • Taghavi P.
      • Chiles K.
      • Mahon P.C.
      • Semenza G.L.
      ) but do suggest that, at least for stretch-induced VEGF expression, the PKC-ζ pathway, independent of Akt activation, predominates within the first several hours in retinal pericytes
      The upstream mechanism by which cellular stretch induces PI 3-kinase and PKC activation in retinal cells is not understood; however, stretch can induce the expression of numerous genes through activation of various intracellular pathways including membrane K+channels, G proteins, intracellular Ca2+, cAMP, cGMP, inositol trisphosphate, protein kinase C, mitogen-activated protein kinase, protein tyrosine kinases, focal adhesion kinase, and alterations in intracellular redox state (
      • Lehoux S.
      • Tedgui A.
      ,
      • Li C.
      • Hu Y.
      • Mayr M.
      • Xu Q.
      ,
      • Hishikawa K.
      • Oemar B.S.
      • Yang Z.
      • Luscher T.F.
      ). Fluid shear stress can also mediate signaling through activation of heterotrimeric and small G proteins, resulting in ERK1/2 and phospholipase C activation with subsequent inositol 1,4,5-trisphosphate and diacylglycerol generation, Ca2+ release, and PKC activation (
      • Ishida T.
      • Takahashi M.
      • Corson M.A.
      • Berk B.C.
      ). However, this mechanism may not be involved in stretch-induced VEGF expression due to the noted ERK1/2 independence and involvement of PKC-ζ, a Ca2+-independent isoform of PKC. Interestingly, mechanical stretch can directly induce growth factor receptor autophosphorylation presumably through changes in cellular morphology leading to altered receptor conformation and subsequent exposure of the kinase domain (
      • Hu Y.
      • Bock G.
      • Wick G.
      • Xu Q.
      ). PDGF receptor can be activated by stretch independently of its ligand. Our data demonstrating stretch increases PDGFR-B tyrosine phosphorylation and subsequent p85 association suggests that such a response may mediate stretch-induced activation of PI 3-kinase. It is as yet unknown whether such stretch-induced receptor activation can mediate VEGF expression.
      Since mechanical stretch can regulate gene expression in a variety of ways (
      • Riser B.L.
      • Ladson-Wofford S.
      • Sharba A.
      • Cortes P.
      • Drake K.
      • Guerin C.J.
      • Yee J.
      • Choi M.E.
      • Segarini P.R.
      • Narins R.G.
      ,
      • Owens G.K.
      ) and since hypertension increases retinal arterial diameter up to 35% (
      • Houben A.J.
      • Canoy M.C.
      • Paling H.A.
      • Derhaag P.J.
      • de Leeuw P.W.
      ,
      • Stanton A.V.
      • Mullaney P.
      • Mee F.
      • O'Brien E.T.
      • O'Malley K.
      ,
      • Rassam S.M.
      • Patel V.
      • Kohner E.M.
      ), it is possible that hypertension-induced stretch in vivo may increase VEGF expression enough to exacerbate ocular conditions characterized by endothelial proliferation and leakage such as diabetic retinopathy. Indeed, retinal expression of VEGF and VEGF-R2 are increased in spontaneously hypertensive rats (
      • Suzuma I.
      • Hata Y.
      • Clermont A.
      • Pokras F.
      • Rook S.L.
      • Suzuma K.
      • Feener E.P.
      • Aiello L.P.
      ). Although the magnitude of stretch experienced by the vasculature is likely to diminish as the internal capillary diameter becomes smaller (
      • Hirschi K.K.
      • D'Amore P.A.
      ), our studies did not identify a maximal VEGF mRNA accumulation as expression continued to increase after all durations of cardiac profile cyclic stretch. Thus, it is possible that even very small increases in cyclic stretch could eventually result in significantly increased VEGF expression.
      This finding may also be important as retinal pericytes are characteristically lost early in the course of diabetic retinopathy (
      • Engerman R.
      • Bloodworth J.M.J.
      • Nelson S.
      ,
      • Cogan D.G.
      • Toussaint D.
      • Kuwabara T.
      ). Thus, even with diminishing numbers, significant localized VEGF expression may be present. Retinal pericytes are an important cell type especially in early stages of retinopathy as they regulate retinal vascular tone and perfusion (
      • Hirschi K.K.
      • D'Amore P.A.
      ), mediate diabetes-induced alterations in autoregulation of retinal blood flow and vasoreactivity (
      • Pourageaud F.
      • De Mey J.G.
      ), and produce VEGF (
      • Aiello L.P.
      • Northrup J.M.
      • Keyt B.A.
      • Takagi H.
      • Iwamoto M.A.
      ). In addition, retinal endothelial cells, which are not compromised until later stages of diabetic retinopathy, respond to stretch with very similar expression of VEGF as do pericytes (
      • Suzuma I.
      • Hata Y.
      • Clermont A.
      • Pokras F.
      • Rook S.L.
      • Suzuma K.
      • Feener E.P.
      • Aiello L.P.
      ). The applicability of these signaling pathways to other cell types remains to be determined.
      In summary, we demonstrate that cardiac profile cyclic stretch induces VEGF expression via PI 3-kinase-mediated activation of PKC-ζ. Furthermore, stretch-induced VEGF expression is independent of ERK1/2, Ras, classical/novel isoforms of PKC, and Akt despite stretch-induced activation of these molecules. In addition, PKC-ζ activation does not mediate ERK1/2 activation. Since each of these molecules has been implicated as mediators of VEGF expression in response to other perturbations, these data suggest that a variety of pathways may be involved in mediating increased VEGF expression in response to diverse stimuli in various cell types. Furthermore, these studies identify new therapeutic targets with potential to ameliorate the well documented clinical exacerbation of ocular diseases, such as diabetic retinopathy, by concomitant hypertension.

      Acknowledgments

      We thank Drs. Masato Kasuga, Yoshimi Takai, Douglas Kirk Ways, and C. Ronald Kahn for providing reagents and technical expertise and Dr. Jerry D. Cavallerano and Pamela Barrows for assistance.

      REFERENCES

        • National Society to Prevent Blindness
        Data Analysis. Vision Problems in the US: Facts and Figures..
        National Society to Prevent Blindness, Schaumburg, IL1980
        • Klein R.
        • Klein B.E.
        • Moss S.E.
        • Cruickshanks K.J.
        Ophthalmology. 1998; 105: 1801-1815
        • Wan N.W.
        • Letchuman R.
        • Noraini N.
        • Ropilah A.R.
        • Zainal M.
        • Ismail I.S.
        • Wan M.W.
        • Faridah I.
        • Singaraveloo M.
        • Sheriff I.H.
        • Khalid B.A.
        Diabetes Res. Clin. Pract. 1999; 46: 213-221
        • Agardh C.D.
        • Agardh E.
        • Torffvit O.
        Diabetes Res. Clin. Pract. 1997; 35: 113-121
        • Aiello L.P.
        • Gardner T.W.
        • King G.L.
        • Blankenship G.W.
        • Cavallerano J.
        • Ferris F.
        • Klein R.
        Diabetes Care. 1998; 21: 143-156
        • Rosenn B.
        • Miodovnik M.
        • Kranias G.
        • Khoury J.
        • Combs C.A.
        • Mimouni F.
        • Siddiqi T.A.
        • Lipman M.J.
        Am. J. Obstet. Gynecol. 1992; 166: 1214-1218
        • Roy M.S.
        Arch. Ophthalmol. 2000; 118: 105-115
        • El-Asrar A.M.
        • Al-Rubeaan K.A.
        • Al-Amro S.A.
        • Kangave D.
        • Moharram O.A.
        Int. Ophthalmol. 1998; 22: 155-161
        • Lopes de Faria J.M.
        • Jalkh A.E.
        • Trempe C.L.
        • McMeel J.W.
        Acta Ophthalmol. Scand. 1999; 77: 170-175
        • UK Prospective Diabetes Study Group
        BMJ. 1998; 317: 703-713
        • Le Floch J.P.
        • Christin S.
        • Bertherat J.
        • Perlemuter L.
        • Hazard J.
        Diabete Metab. 1990; 16: 26-29
        • Tso M.O.
        • Jampol L.M.
        Ophthalmology. 1982; 89: 1132-1145
        • Macular Photocoagulation Study Group
        Arch. Ophthalmol. 1997; 115: 741-747
        • Shweiki D.
        • Itin A.
        • Soffer D.
        • Keshet E.
        Nature. 1992; 359: 843-845
        • Shweiki D.
        • Itin A.
        • Neufeld G.
        • Gitay-Goren H.
        • Keshet E.
        J. Clin. Invest. 1993; 91: 2235-2243
        • Shweiki D.
        • Neeman M.
        • Itin A.
        • Keshet E.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 768-772
        • Senger D.R.
        • Connolly D.T.
        • Van de Water L.
        • Feder J.
        • Dvorak H.F.
        Cancer Res. 1990; 50: 1774-1778
        • Aiello L.P.
        • Avery R.L.
        • Arrigg P.G.
        • Keyt B.A.
        • Jampel H.D.
        • Shah S.T.
        • Pasquale L.R.
        • Thieme H.
        • Iwamoto M.A.
        • Park J.E.
        N. Engl. J. Med. 1994; 331: 1480-1487
        • Aiello L.P.
        • Northrup J.M.
        • Keyt B.A.
        • Takagi H.
        • Iwamoto M.A.
        Arch. Ophthalmol. 1995; 113: 1538-1544
        • Pierce E.A.
        • Avery R.L.
        • Foley E.D.
        • Aiello L.P.
        • Smith L.E.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 905-909
        • Simorre-Pinatel V.
        • Guerrin M.
        • Chollet P.
        • Penary M.
        • Clamens S.
        • Malecaze F.
        • Plouet J.
        Investig. Ophthalmol. Vis. Sci. 1994; 35: 3393-3400
        • Adamis A.P.
        • Shima D.T.
        • Yeo K.T.
        • Yeo T.K.
        • Brown L.F.
        • Berse B.
        • D'Amore P.A.
        • Folkman J.
        Biochem. Biophys. Res. Commun. 1993; 193: 631-638
        • Frank R.N.
        Ophthalmic Res. 1997; 29: 341-353
        • Amin R.H.
        • Frank R.N.
        • Kennedy A.
        • Eliott D.
        • Puklin J.E.
        • Abrams G.W.
        Investig. Ophthalmol. Vis. Sci. 1997; 38: 36-47
        • Ishibashi T.
        • Hata Y.
        • Yoshikawa H.
        • Nakagawa K.
        • Sueishi K.
        • Inomata H.
        Graefe's Arch. Clin. Exp. Ophthalmol. 1997; 235: 159-167
        • Kvanta A.
        • Algvere P.V.
        • Berglin L.
        • Seregard S.
        Investig. Ophthalmol. Vis. Sci. 1996; 37: 1929-1934
        • Pe'er J.
        • Folberg R.
        • Itin A.
        • Gnessin H.
        • Hemo I.
        • Keshet E.
        Ophthalmology. 1998; 105: 412-416
        • Fong G.H.
        • Rossant J.
        • Gertsenstein M.
        • Breitman M.L.
        Nature. 1995; 376: 66-70
        • Shalaby F.
        • Rossant J.
        • Yamaguchi T.P.
        • Gertsenstein M.
        • Wu X.F.
        • Breitman M.L.
        • Schuh A.C.
        Nature. 1995; 376: 62-66
        • Safar M.E.
        • Peronneau P.A.
        • Levenson J.A.
        • Toto-Moukouo J.A.
        • Simon A.C.
        Circulation. 1981; 63: 393-400
        • Houben A.J.
        • Canoy M.C.
        • Paling H.A.
        • Derhaag P.J.
        • de Leeuw P.W.
        J. Hypertens. 1995; 13: 1729-1733
        • Li J.
        • Hampton T.
        • Morgan J.P.
        • Simons M.
        J. Clin. Invest. 1997; 100: 18-24
        • Seko Y.
        • Takahashi N.
        • Shibuya M.
        • Yazaki Y.
        Biochem. Biophys. Res. Commun. 1999; 254: 462-465
        • Gruden G.
        • Thomas S.
        • Burt D.
        • Lane S.
        • Chusney G.
        • Sacks S.
        • Viberti G.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12112-12116
        • Seko Y.
        • Fujikura H.
        • Pang J.
        • Tokoro T.
        • Shimokawa H.
        Investig. Ophthalmol. Vis. Sci. 1999; 40: 3287-3291
        • Suzuma I.
        • Hata Y.
        • Clermont A.
        • Pokras F.
        • Rook S.L.
        • Suzuma K.
        • Feener E.P.
        • Aiello L.P.
        Diabetes. 2001; 50: 444-454
        • Ishida T.
        • Takahashi M.
        • Corson M.A.
        • Berk B.C.
        Ann. N. Y. Acad. Sci. 1997; 811: 12-23
        • Sadoshima J.
        • Izumo S.
        EMBO J. 1993; 12: 1681-1692
        • Ziegler T.
        • Silacci P.
        • Harrison V.J.
        • Hayoz D.
        Hypertension. 1998; 32: 351-355
        • Yamazaki T.
        • Komuro I.
        • Kudoh S.
        • Zou Y.
        • Shiojima I.
        • Hiroi Y.
        • Mizuno T.
        • Maemura K.
        • Kurihara H.
        • Aikawa R.
        • Takano H.
        • Yazaki Y.
        J. Biol. Chem. 1996; 271: 3221-3228
        • Hu Y.
        • Bock G.
        • Wick G.
        • Xu Q.
        FASEB J. 1998; 12: 1135-1142
        • Park J.M.
        • Borer J.G.
        • Freeman M.R.
        • Peters C.A.
        Am. J. Physiol. 1998; 275: C1247-C1254
        • Marrero M.B.
        • Schieffer B.
        • Paxton W.G.
        • Heerdt L.
        • Berk B.C.
        • Delafontaine P.
        • Bernstein K.E.
        Nature. 1995; 375: 247-250
        • Tamura K.
        • Umemura S.
        • Nyui N.
        • Hibi K.
        • Ishigami T.
        • Kihara M.
        • Toya Y.
        • Ishii M.
        Am. J. Physiol. 1998; 275: R1-R9
        • Persson K.
        • Sando J.J.
        • Tuttle J.B.
        • Steers W.D.
        Am. J. Physiol. 1995; 269: C1018-C1024
        • Koya D.
        • King G.L.
        Diabetes. 1998; 47: 859-866
        • Bursell S.E.
        • Takagi C.
        • Clermont A.C.
        • Takagi H.
        • Mori F.
        • Ishii H.
        • King G.L.
        Investig. Ophthalmol. Vis. Sci. 1997; 38: 2711-2720
        • King G.L.
        • Ishii H.
        • Koya D.
        Kidney Int. Suppl. 1997; 60: S77-S85
        • Pal S.
        • Claffey K.P.
        • Cohen H.T.
        • Mukhopadhyay D.
        J. Biol. Chem. 1998; 273: 26277-26280
        • Pal S.
        • Claffey K.P.
        • Dvorak H.F.
        • Mukhopadhyay D.
        J. Biol. Chem. 1997; 272: 27509-27512
        • Ono Y.
        • Fujii T.
        • Ogita K.
        • Kikkawa U.
        • Igarashi K.
        • Nishizuka Y.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103
        • Nakanishi H.
        • Exton J.H.
        J. Biol. Chem. 1992; 267: 16347-16354
        • Nakanishi H.
        • Brewer K.A.
        • Exton J.H.
        J. Biol. Chem. 1993; 268: 13-16
        • Berra E.
        • Diaz-Meco M.T.
        • Dominguez I.
        • Municio M.M.
        • Sanz L.
        • Lozano J.
        • Chapkin R.S.
        • Moscat J.
        Cell. 1993; 74: 555-563
        • Chou M.M.
        • Hou W.
        • Johnson J.
        • Graham L.K.
        • Lee M.H.
        • Chen C.S.
        • Newton A.C.
        • Schaffhausen B.S.
        • Toker A.
        Curr. Biol. 1998; 8: 1069-1077
        • Pal S.
        • Datta K.
        • Khosravi-Far R.
        • Mukhopadhyay D.
        J. Biol. Chem. 2001; 276: 2395-2403
        • Shih S.C.
        • Mullen A.
        • Abrams K.
        • Mukhopadhyay D.
        • Claffey K.P.
        J. Biol. Chem. 1999; 274: 15407-15414
        • Wellner M.
        • Maasch C.
        • Kupprion C.
        • Lindschau C.
        • Luft F.C.
        • Haller H.
        Arterioscler. Thromb. Vasc. Biol. 1999; 19: 178-185
        • King G.L.
        • Goodman A.D.
        • Buzney S.
        • Moses A.
        • Kahn C.R.
        J. Clin. Invest. 1985; 75: 1028-1036
        • Nayak R.C.
        • Berman A.B.
        • George K.L.
        • Eisenbarth G.S.
        • King G.L.
        J. Cell Biol. 1987; 105: 1595-1601
        • Burgering B.M.
        • Coffer P.J.
        Nature. 1995; 376: 599-602
        • Kitamura T.
        • Ogawa W.
        • Sakaue H.
        • Hino Y.
        • Kuroda S.
        • Takata M.
        • Matsumoto M.
        • Maeda T.
        • Konishi H.
        • Kikkawa U.
        • Kasuga M.
        Mol. Cell. Biol. 1998; 18: 3708-3717
        • Ueki K.
        • Matsuda S.
        • Tobe K.
        • Gotoh Y.
        • Tamemoto H.
        • Yachi M.
        • Akanuma Y.
        • Yazaki Y.
        • Nishida E.
        • Kadowaki T.
        J. Biol. Chem. 1994; 269: 15756-15761
        • Her J.H.
        • Lakhani S.
        • Zu K.
        • Vila J.
        • Dent P.
        • Sturgill T.W.
        • Weber M.J.
        Biochem. J. 1993; 296: 25-31
        • Ueki K.
        • Yamamoto-Honda R.
        • Kaburagi Y.
        • Yamauchi T.
        • Tobe K.
        • Burgering B.M.
        • Coffer P.J.
        • Komuro I.
        • Akanuma Y.
        • Yazaki Y.
        • Kadowaki T.
        J. Biol. Chem. 1998; 273: 5315-5322
        • Hara K.
        • Yonezawa K.
        • Sakaue H.
        • Ando A.
        • Kotani K.
        • Kitamura T.
        • Kitamura Y.
        • Ueda H.
        • Stephens L.
        • Jackson T.R.
        • et al.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419
        • Uberall F.
        • Hellbert K.
        • Kampfer S.
        • Maly K.
        • Villunger A.
        • Spitaler M.
        • Mwanjewe J.
        • Baier-Bitterlich G.
        • Baier G.
        • Grunicke H.H.
        J. Cell Biol. 1999; 144: 413-425
        • He T.C.
        • Zhou S.
        • da Costa L.T.
        • Yu J.
        • Kinzler K.W.
        • Vogelstein B.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514
        • Miyake S.
        • Makimura M.
        • Kanegae Y.
        • Harada S.
        • Sato Y.
        • Takamori K.
        • Tokuda C.
        • Saito I.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324
        • Xia P.
        • Aiello L.P.
        • Ishii H.
        • Jiang Z.Y.
        • Park D.J.
        • Robinson G.S.
        • Takagi H.
        • Newsome W.P.
        • Jirousek M.R.
        • King G.L.
        J. Clin. Invest. 1996; 98: 2018-2026
        • Takeda H.
        • Matozaki T.
        • Takada T.
        • Noguchi T.
        • Yamao T.
        • Tsuda M.
        • Ochi F.
        • Fukunaga K.
        • Inagaki K.
        • Kasuga M.
        EMBO J. 1999; 18: 386-395
        • Akimoto K.
        • Takahashi R.
        • Moriya S.
        • Nishioka N.
        • Takayanagi J.
        • Kimura K.
        • Fukui Y.
        • Osada S.
        • Mizuno K.
        • Hirai S.
        • Kazlauskas A.
        • Ohno S.
        EMBO J. 1996; 15: 788-798
        • Jung Y.D.
        • Nakano K.
        • Liu W.
        • Gallick G.E.
        • Ellis L.M.
        Cancer Res. 1999; 59: 4804-4807
        • Okajima E.
        • Thorgeirsson U.P.
        Biochem. Biophys. Res. Commun. 2000; 270: 108-111
        • Engerman R.
        • Bloodworth J.M.J.
        • Nelson S.
        Diabetes. 1977; 26: 760-769
        • Pedram A.
        • Razandi M.
        • Hu R.M.
        • Levin E.R.
        J. Biol. Chem. 1997; 272: 17097-17103
        • Mukhopadhyay D.
        • Tsiokas L.
        • Zhou X.M.
        • Foster D.
        • Brugge J.S.
        • Sukhatme V.P.
        Nature. 1995; 375: 577-581
        • Milanini J.
        • Vinals F.
        • Pouyssegur J.
        • Pages G.
        J. Biol. Chem. 1998; 273: 18165-18172
        • Standaert M.L.
        • Galloway L.
        • Karnam P.
        • Bandyopadhyay G.
        • Moscat J.
        • Farese R.V.
        J. Biol. Chem. 1997; 272: 30075-30082
        • Neri L.M.
        • Martelli A.M.
        • Borgatti P.
        • Colamussi M.L.
        • Marchisio M.
        • Capitani S.
        FASEB J. 1999; 13: 2299-2310
        • Mizukami Y.
        • Kobayashi S.
        • Uberall F.
        • Hellbert K.
        • Kobayashi N.
        • Yoshida K.
        J. Biol. Chem. 2000; 275: 19921-19927
        • Sajan M.P.
        • Standaert M.L.
        • Bandyopadhyay G.
        • Quon M.J.
        • Burke Jr., T.R.
        • Farese R.V.
        J. Biol. Chem. 1999; 274: 30495-30500
        • Pourageaud F.
        • De Mey J.G.
        Am. J. Physiol. 1998; 274: H1301-H1307
        • Monick M.M.
        • Carter A.B.
        • Flaherty D.M.
        • Peterson M.W.
        • Hunninghake G.W.
        J. Immunol. 2000; 165: 4632-4639
        • Jiang B.H.
        • Zheng J.Z.
        • Aoki M.
        • Vogt P.K.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1749-1753
        • Laughner E.
        • Taghavi P.
        • Chiles K.
        • Mahon P.C.
        • Semenza G.L.
        Mol. Cell. Biol. 2001; 21: 3995-4004
        • Lehoux S.
        • Tedgui A.
        Hypertension. 1998; 32: 338-345
        • Li C.
        • Hu Y.
        • Mayr M.
        • Xu Q.
        J. Biol. Chem. 1999; 274: 25273-25280
        • Hishikawa K.
        • Oemar B.S.
        • Yang Z.
        • Luscher T.F.
        Circ. Res. 1997; 81: 797-803
        • Riser B.L.
        • Ladson-Wofford S.
        • Sharba A.
        • Cortes P.
        • Drake K.
        • Guerin C.J.
        • Yee J.
        • Choi M.E.
        • Segarini P.R.
        • Narins R.G.
        Kidney Int. 1999; 56: 428-439
        • Owens G.K.
        Am. J. Physiol. 1989; 257: H1755-H1765
        • Stanton A.V.
        • Mullaney P.
        • Mee F.
        • O'Brien E.T.
        • O'Malley K.
        J. Hypertens. 1995; 13: 41-48
        • Rassam S.M.
        • Patel V.
        • Kohner E.M.
        Exp. Physiol. 1995; 80: 53-68
        • Hirschi K.K.
        • D'Amore P.A.
        Cardiovasc. Res. 1996; 32: 687-698
        • Cogan D.G.
        • Toussaint D.
        • Kuwabara T.
        Arch. Ophthalmol. 1961; 66: 366-378