8fdcf574c3ec83091f98dca2aa6637ef 271474.pdf 3a95a1c34afc57600b260daf1ff051e35bc5d765 271474.pdf c7af665ddcd60d4aa3978404b90e20f587f89bc5bcdf56a4cf8b2225d8a3f513 271474.pdf Title: The Interplay of Mitochondrial Oxidative Stress and Endoplasmic Reticulum Stress in Cardiovascular Fibrosis in Obese Rats Subject: We have evaluated the role of mitochondrial oxidative stress and its association with endoplasmic reticulum (ER) stress activation in the progression of obesity-related cardiovascular fibrosis. MitoQ (200 M) was orally administered for 7 weeks to male Wistar rats that were fed a high-fat diet (HFD, 35% fat) or a control diet (CT, 3.5% fat). Obese animals presented cardiovascular fibrosis accompanied by increased levels of extracellular matrix proteins and profibrotic mediators. These alterations were associated with ER stress activation characterized by enhanced levels (in heart and aorta vs. CT group, respectively) of immunoglobulin binding protein (BiP; 2.1-and 2.6-fold, respectively), protein disulfide-isomerase A6 (PDIA6; 1.9-fold) and CCAAT-enhancer-binding homologous protein (CHOP; 1.5- and 1.8-fold, respectively). MitoQ treatment was able to prevent (p < 0.05) these modifications at cardiac and aortic levels. MitoQ (5 nM) and the ER stress inhibitor, 4-phenyl butyric acid (4 M), were able to block the prooxidant and profibrotic effects of angiotensin II (Ang II, 10-6 M) in cardiac and vascular cells. Therefore, the data show a crosstalk between mitochondrial oxidative stress and ER stress activation, which mediates the development of cardiovascular fibrosis in the context of obesity and in which Ang II can play a relevant role. Keywords: cardiovascular fibrosis; endoplasmic reticulum stress; mitochondrial oxidative stress; obesity Author: Francisco V. Souza-Neto, Sara Jiménez-González, Beatriz Delgado-Valero, Raquel Jurado-López, Marie Genty, Ana Romero-Miranda, Cristina Rodríguez, María Luisa Nieto, Ernesto Martínez-Martínez and Victoria Cachofeiro Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Aug 11 12:19:01 2021 CEST ModDate: Wed Aug 11 12:49:26 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2501033 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JYPLJJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 OPZDHV+VnURWPalladioL Type 1 Custom yes yes no 15 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 19 0 TIOBLC+URWPalladioL-Ital Type 1 Custom yes yes no 23 0 VVVOOL+URWPalladioL-Bold Type 1 Custom yes yes no 28 0 ESQAIB+URWPalladioL-Roma Type 1 Custom yes yes no 32 0 QMDDXF+CMSY10 Type 1 Builtin yes yes no 36 0 KUDXZB+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 44 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 66 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 70 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 74 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 92 0 IDOFNF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 110 0 IDOFNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 113 0 IDOFNH+PalatinoLinotype TrueType WinAnsi yes yes no 119 0 IDOFNJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 122 0 IDOFNF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 140 0 IDOFNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 143 0 IDOFNH+PalatinoLinotype TrueType WinAnsi yes yes no 149 0 IDOFNJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 152 0 IDOFNF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 167 0 IDOFNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 170 0 IDOFNH+PalatinoLinotype TrueType WinAnsi yes yes no 176 0 IDOFNJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 179 0 IDOFNF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 194 0 IDOFNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 197 0 IDOFNH+PalatinoLinotype TrueType WinAnsi yes yes no 203 0 IDOFNJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 206 0 IDOFNF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 220 0 IDOFNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 223 0 IDOFNH+PalatinoLinotype TrueType WinAnsi yes yes no 229 0 IDOFNJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 232 0 IDOFNF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 247 0 IDOFNG+PalatinoLinotype CID TrueType Identity-H yes yes yes 250 0 IDOFNH+PalatinoLinotype TrueType WinAnsi yes yes no 256 0 IDOFNJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 259 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 03:06:26 CEST RepresentationInformation: 271474.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 12:05:06 CEST Size: 2501033 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 527 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Animal Model Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Histological Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Cell Isolation and Cell Culture Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Western Blot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Retrotranscription and Real-Time PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Measurement of Intracellular Superoxide Anion Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: Mitochondrial Oxidative Stress Promotes ECM Protein Deposition and ER Stress Activation at Cardiac Level in Obesity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: MitoQ Treatment Improves Vascular Fibrosis and ER Stress Activation in the Aorta of HFD Animals Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Mitochondrial Oxidative Stress Mediates the Effects of Ang II in Cardiac Fibroblasts Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Mitochondrial Oxidative Stress Mediates the Effects of Ang II in Vascular Smooth Muscle Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Inhibition of Endoplasmic Reticulum Stress Blocks the Prooxidant and Profibrotic Effects of Ang II in Cardiovascular Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Limitations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Info: Title: The Interplay of Mitochondrial Oxidative Stress and Endoplasmic Reticulum Stress in Cardiovascular Fibrosis in Obese Rats Author: Francisco V. Souza-Neto, Sara Jiménez-González, Beatriz Delgado-Valero, Raquel Jurado-López, Marie Genty, Ana Romero-Miranda, Cristina Rodríguez, María Luisa Nieto, Ernesto Martínez-Martínez and Victoria Cachofeiro Subject: We have evaluated the role of mitochondrial oxidative stress and its association with endoplasmic reticulum (ER) stress activation in the progression of obesity-related cardiovascular fibrosis. MitoQ (200 M) was orally administered for 7 weeks to male Wistar rats that were fed a high-fat diet (HFD, 35% fat) or a control diet (CT, 3.5% fat). Obese animals presented cardiovascular fibrosis accompanied by increased levels of extracellular matrix proteins and profibrotic mediators. These alterations were associated with ER stress activation characterized by enhanced levels (in heart and aorta vs. CT group, respectively) of immunoglobulin binding protein (BiP; 2.1-and 2.6-fold, respectively), protein disulfide-isomerase A6 (PDIA6; 1.9-fold) and CCAAT-enhancer-binding homologous protein (CHOP; 1.5- and 1.8-fold, respectively). MitoQ treatment was able to prevent (p < 0.05) these modifications at cardiac and aortic levels. MitoQ (5 nM) and the ER stress inhibitor, 4-phenyl butyric acid (4 M), were able to block the prooxidant and profibrotic effects of angiotensin II (Ang II, 10-6 M) in cardiac and vascular cells. Therefore, the data show a crosstalk between mitochondrial oxidative stress and ER stress activation, which mediates the development of cardiovascular fibrosis in the context of obesity and in which Ang II can play a relevant role. Keywords: cardiovascular fibrosis; endoplasmic reticulum stress; mitochondrial oxidative stress; obesity Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Aug 11 12:19:01 CEST 2021 ModDate: Wed Aug 11 12:49:26 CEST 2021 ID: 0x66ae2b5fd4b3ec161a7e1661d58a79ea, 0x66ae2b5fd4b3ec161a7e1661d58a79ea Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: ESQAIB+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 243 FontDescriptor: FontName: ESQAIB+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: QMDDXF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: QMDDXF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: TIOBLC+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: TIOBLC+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JYPLJJ+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: JYPLJJ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVVOOL+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 135 FontDescriptor: FontName: VVVOOL+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: OPZDHV+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: OPZDHV+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 175, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 282, 643, 293, 656 Annotation: Subtype: Link Rect: 409, 643, 420, 656 Annotation: Subtype: Link Rect: 99, 630, 110, 644 Annotation: Subtype: Link Rect: 220, 630, 231, 644 Annotation: Subtype: Link Rect: 341, 630, 352, 644 Annotation: Subtype: Link Rect: 451, 630, 462, 644 Annotation: Subtype: Link Rect: 185, 620, 196, 631 Annotation: Subtype: Link Rect: 331, 620, 342, 631 Annotation: Subtype: Link Rect: 121, 346, 154, 355 Annotation: Subtype: Link Rect: 35, 334, 137, 343 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 301, 155, 308, 164 ActionDest: 271 Annotation: Subtype: Link Rect: 507, 118, 514, 127 ActionDest: 271 Annotation: Subtype: Link Rect: 213, 92, 220, 102 ActionDest: 271 Annotation: Subtype: Link Rect: 407, 68, 414, 76 ActionDest: 271 Annotation: Subtype: Link Rect: 417, 67, 424, 76 ActionDest: 274 Annotation: Subtype: Link Rect: 131, 17, 277, 28 Annotation: Subtype: Link Rect: 390, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 470, 87, 489 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 385, 724, 392, 733 ActionDest: 274 Annotation: Subtype: Link Rect: 225, 686, 232, 695 ActionDest: 274 Annotation: Subtype: Link Rect: 232, 686, 239, 695 ActionDest: 274 Annotation: Subtype: Link Rect: 333, 661, 345, 670 ActionDest: 274 Annotation: Subtype: Link Rect: 345, 661, 357, 670 ActionDest: 274 Annotation: Subtype: Link Rect: 465, 661, 477, 670 ActionDest: 274 Annotation: Subtype: Link Rect: 352, 586, 359, 595 ActionDest: 274 Annotation: Subtype: Link Rect: 313, 536, 325, 545 ActionDest: 274 Annotation: Subtype: Link Rect: 325, 536, 337, 545 ActionDest: 274 Annotation: Subtype: Link Rect: 308, 523, 320, 532 ActionDest: 274 Annotation: Subtype: Link Rect: 543, 523, 555, 532 ActionDest: 274 Annotation: Subtype: Link Rect: 235, 511, 247, 520 ActionDest: 274 Annotation: Subtype: Link Rect: 316, 511, 328, 520 ActionDest: 274 Annotation: Subtype: Link Rect: 440, 511, 452, 520 ActionDest: 274 Annotation: Subtype: Link Rect: 374, 498, 386, 507 ActionDest: 274 Annotation: Subtype: Link Rect: 387, 498, 399, 507 ActionDest: 274 Annotation: Subtype: Link Rect: 347, 473, 358, 482 ActionDest: 274 Annotation: Subtype: Link Rect: 355, 423, 366, 432 ActionDest: 274 Annotation: Subtype: Link Rect: 422, 222, 434, 231 ActionDest: 274 Annotation: Subtype: Link Rect: 544, 197, 556, 206 ActionDest: 274 Annotation: Subtype: Link Rect: 427, 159, 439, 168 ActionDest: 274 Annotation: Subtype: Link Rect: 517, 122, 529, 131 ActionDest: 274 Annotation: Subtype: Link Rect: 530, 121, 542, 131 ActionDest: 274 Annotation: Subtype: Link Rect: 239, 96, 251, 105 ActionDest: 277 Annotation: Subtype: Link Rect: 410, 84, 422, 93 ActionDest: 277 Annotation: Subtype: Link Rect: 423, 84, 435, 93 ActionDest: 277 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 358, 710, 370, 719 ActionDest: 277 Annotation: Subtype: Link Rect: 544, 495, 556, 504 ActionDest: 277 Annotation: Subtype: Link Rect: 333, 218, 340, 227 ActionDest: 274 Annotation: Subtype: Link Rect: 340, 218, 352, 227 ActionDest: 277 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 321, 707, 328, 716 ActionDest: 274 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 198, 732, 205, 744 ActionDest: 96 Annotation: Subtype: Link Rect: 529, 720, 536, 732 ActionDest: 96 Annotation: Subtype: Link Rect: 352, 294, 359, 306 ActionDest: 96 Annotation: Subtype: Link Rect: 473, 269, 480, 281 ActionDest: 96 Annotation: Subtype: Link Rect: 343, 244, 350, 256 ActionDest: 96 Annotation: Subtype: Link Rect: 534, 120, 541, 132 ActionDest: 128 Annotation: Subtype: Link Rect: 255, 95, 262, 107 ActionDest: 128 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 352, 682, 359, 694 ActionDest: 128 Annotation: Subtype: Link Rect: 389, 250, 396, 262 ActionDest: 128 Annotation: Subtype: Link Rect: 201, 237, 208, 249 ActionDest: 128 Annotation: Subtype: Link Rect: 483, 237, 490, 249 ActionDest: 128 Annotation: Subtype: Link Rect: 423, 212, 430, 224 ActionDest: 128 Annotation: Subtype: Link Rect: 515, 124, 522, 136 ActionDest: 157 Annotation: Subtype: Link Rect: 406, 99, 413, 111 ActionDest: 157 Annotation: Subtype: Link Rect: 517, 86, 524, 98 ActionDest: 157 Annotation: Subtype: Link Rect: 532, 61, 539, 73 ActionDest: 157 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 541, 306, 548, 317 ActionDest: 157 Annotation: Subtype: Link Rect: 410, 281, 417, 293 ActionDest: 157 Annotation: Subtype: Link Rect: 222, 243, 229, 255 ActionDest: 157 Annotation: Subtype: Link Rect: 251, 144, 258, 156 ActionDest: 184 Annotation: Subtype: Link Rect: 262, 132, 269, 144 ActionDest: 184 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 338, 321, 345, 333 ActionDest: 184 Annotation: Subtype: Link Rect: 337, 296, 344, 308 ActionDest: 184 Annotation: Subtype: Link Rect: 503, 283, 510, 295 ActionDest: 184 Annotation: Subtype: Link Rect: 355, 197, 362, 209 ActionDest: 210 Annotation: Subtype: Link Rect: 248, 147, 255, 159 ActionDest: 210 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 507, 318, 514, 330 ActionDest: 210 Annotation: Subtype: Link Rect: 311, 293, 318, 305 ActionDest: 210 Annotation: Subtype: Link Rect: 254, 268, 261, 281 ActionDest: 237 Annotation: Subtype: Link Rect: 407, 268, 414, 281 ActionDest: 237 Annotation: Subtype: Link Rect: 200, 230, 207, 242 ActionDest: 237 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 443, 120, 455, 129 ActionDest: 277 Annotation: Subtype: Link Rect: 456, 120, 468, 129 ActionDest: 277 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 519, 722, 531, 731 ActionDest: 274 Annotation: Subtype: Link Rect: 531, 722, 543, 731 ActionDest: 277 Annotation: Subtype: Link Rect: 544, 722, 556, 731 ActionDest: 277 Annotation: Subtype: Link Rect: 227, 685, 239, 694 ActionDest: 277 Annotation: Subtype: Link Rect: 240, 685, 252, 694 ActionDest: 277 Annotation: Subtype: Link Rect: 262, 634, 274, 643 ActionDest: 277 Annotation: Subtype: Link Rect: 375, 609, 387, 618 ActionDest: 277 Annotation: Subtype: Link Rect: 488, 609, 500, 618 ActionDest: 277 Annotation: Subtype: Link Rect: 196, 559, 208, 568 ActionDest: 277 Annotation: Subtype: Link Rect: 490, 546, 502, 556 ActionDest: 277 Annotation: Subtype: Link Rect: 382, 509, 389, 518 ActionDest: 274 Annotation: Subtype: Link Rect: 269, 459, 281, 468 ActionDest: 277 Annotation: Subtype: Link Rect: 493, 434, 505, 443 ActionDest: 277 Annotation: Subtype: Link Rect: 380, 408, 392, 417 ActionDest: 277 Annotation: Subtype: Link Rect: 393, 408, 405, 417 ActionDest: 277 Annotation: Subtype: Link Rect: 362, 346, 374, 355 ActionDest: 274 Annotation: Subtype: Link Rect: 293, 320, 305, 330 ActionDest: 277 Annotation: Subtype: Link Rect: 308, 320, 320, 330 ActionDest: 280 Annotation: Subtype: Link Rect: 529, 233, 541, 242 ActionDest: 280 Annotation: Subtype: Link Rect: 544, 233, 556, 242 ActionDest: 280 Annotation: Subtype: Link Rect: 280, 120, 292, 129 ActionDest: 277 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 407, 710, 419, 719 ActionDest: 280 Annotation: Subtype: Link Rect: 496, 672, 508, 681 ActionDest: 280 Annotation: Subtype: Link Rect: 216, 634, 228, 643 ActionDest: 280 Annotation: Subtype: Link Rect: 448, 597, 460, 606 ActionDest: 280 Annotation: Subtype: Link Rect: 482, 584, 494, 593 ActionDest: 280 Annotation: Subtype: Link Rect: 495, 584, 506, 593 ActionDest: 280 Annotation: Subtype: Link Rect: 243, 572, 255, 581 ActionDest: 280 Annotation: Subtype: Link Rect: 543, 459, 555, 468 ActionDest: 280 Annotation: Subtype: Link Rect: 230, 446, 242, 455 ActionDest: 280 Annotation: Subtype: Link Rect: 364, 446, 376, 455 ActionDest: 280 Annotation: Subtype: Link Rect: 479, 358, 491, 367 ActionDest: 277 Annotation: Subtype: Link Rect: 301, 233, 313, 242 ActionDest: 280 Annotation: Subtype: Link Rect: 313, 233, 325, 242 ActionDest: 280 Annotation: Subtype: Link Rect: 445, 82, 457, 91 ActionDest: 277 Annotation: Subtype: Link Rect: 457, 82, 469, 91 ActionDest: 280 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 425, 583, 560, 594 Annotation: Subtype: Link Rect: 166, 571, 274, 582 Annotation: Subtype: Link Rect: 519, 139, 557, 150 Annotation: Subtype: Link Rect: 59, 128, 95, 138 Annotation: Subtype: Link Rect: 104, 105, 142, 115 Annotation: Subtype: Link Rect: 148, 105, 184, 115 Annotation: Subtype: Link Rect: 520, 81, 557, 92 Annotation: Subtype: Link Rect: 401, 58, 439, 69 Annotation: Subtype: Link Rect: 445, 58, 481, 69 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 354, 709, 392, 720 Annotation: Subtype: Link Rect: 398, 709, 434, 720 Annotation: Subtype: Link Rect: 272, 686, 309, 697 Annotation: Subtype: Link Rect: 408, 663, 446, 674 Annotation: Subtype: Link Rect: 119, 641, 157, 651 Annotation: Subtype: Link Rect: 163, 641, 199, 651 Annotation: Subtype: Link Rect: 339, 606, 376, 617 Annotation: Subtype: Link Rect: 325, 571, 363, 582 Annotation: Subtype: Link Rect: 298, 537, 335, 548 Annotation: Subtype: Link Rect: 246, 502, 283, 513 Annotation: Subtype: Link Rect: 290, 502, 326, 513 Annotation: Subtype: Link Rect: 516, 491, 553, 502 Annotation: Subtype: Link Rect: 59, 469, 97, 479 Annotation: Subtype: Link Rect: 298, 445, 336, 456 Annotation: Subtype: Link Rect: 488, 433, 524, 444 Annotation: Subtype: Link Rect: 369, 410, 407, 421 Annotation: Subtype: Link Rect: 335, 387, 372, 398 Annotation: Subtype: Link Rect: 378, 387, 414, 398 Annotation: Subtype: Link Rect: 95, 354, 133, 364 Annotation: Subtype: Link Rect: 59, 331, 97, 341 Annotation: Subtype: Link Rect: 109, 308, 146, 318 Annotation: Subtype: Link Rect: 59, 273, 97, 283 Annotation: Subtype: Link Rect: 103, 273, 139, 283 Annotation: Subtype: Link Rect: 470, 238, 507, 249 Annotation: Subtype: Link Rect: 127, 204, 164, 214 Annotation: Subtype: Link Rect: 171, 204, 207, 214 Annotation: Subtype: Link Rect: 379, 180, 416, 191 Annotation: Subtype: Link Rect: 429, 157, 467, 168 Annotation: Subtype: Link Rect: 473, 157, 509, 168 Annotation: Subtype: Link Rect: 188, 123, 226, 134 Annotation: Subtype: Link Rect: 232, 123, 268, 134 Annotation: Subtype: Link Rect: 131, 89, 169, 99 Annotation: Subtype: Link Rect: 175, 89, 211, 99 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 104, 710, 141, 720 Annotation: Subtype: Link Rect: 492, 686, 529, 697 Annotation: Subtype: Link Rect: 240, 652, 278, 663 Annotation: Subtype: Link Rect: 95, 630, 133, 640 Annotation: Subtype: Link Rect: 139, 630, 175, 640 Annotation: Subtype: Link Rect: 479, 606, 515, 617 Annotation: Subtype: Link Rect: 521, 606, 557, 617 Annotation: Subtype: Link Rect: 59, 572, 97, 582 Annotation: Subtype: Link Rect: 103, 572, 139, 582 Annotation: Subtype: Link Rect: 457, 548, 495, 559 Annotation: Subtype: Link Rect: 280, 525, 318, 536 Annotation: Subtype: Link Rect: 182, 491, 220, 502 Annotation: Subtype: Link Rect: 503, 456, 540, 467 Annotation: Subtype: Link Rect: 95, 423, 132, 433 Annotation: Subtype: Link Rect: 59, 400, 97, 410 Annotation: Subtype: Link Rect: 59, 365, 97, 375 Annotation: Subtype: Link Rect: 487, 341, 524, 352 Annotation: Subtype: Link Rect: 182, 307, 220, 318 Annotation: Subtype: Link Rect: 95, 273, 131, 283 Annotation: Subtype: Link Rect: 416, 238, 454, 249 Annotation: Subtype: Link Rect: 460, 238, 496, 249 Annotation: Subtype: Link Rect: 316, 215, 353, 226 Annotation: Subtype: Link Rect: 360, 215, 396, 226 Annotation: Subtype: Link Rect: 299, 192, 336, 203 Annotation: Subtype: Link Rect: 268, 169, 304, 180 Annotation: Subtype: Link Rect: 59, 135, 97, 145 Annotation: Subtype: Link Rect: 497, 123, 535, 134 Annotation: Subtype: Link Rect: 189, 100, 227, 111 Annotation: Subtype: Link Rect: 86, 66, 123, 76 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 181, 722, 219, 732 Annotation: Subtype: Link Rect: 232, 699, 270, 709 Annotation: Subtype: Link Rect: 276, 699, 312, 709 Annotation: Subtype: Link Rect: 519, 675, 557, 686 Annotation: Subtype: Link Rect: 59, 664, 95, 674 Annotation: Subtype: Link Rect: 271, 641, 309, 651 Annotation: Subtype: Link Rect: 315, 641, 351, 651 Annotation: Subtype: Link Rect: 221, 617, 258, 628 Annotation: Subtype: Link Rect: 255, 548, 293, 559 Annotation: Subtype: Link Rect: 377, 525, 414, 536 Annotation: Subtype: Link Rect: 59, 492, 97, 502 Annotation: Subtype: Link Rect: 103, 492, 139, 502 Annotation: Subtype: Link Rect: 140, 469, 178, 479 Annotation: Subtype: Link Rect: 250, 433, 288, 444 Annotation: Subtype: Link Rect: 407, 410, 445, 421 Annotation: Subtype: Link Rect: 451, 410, 487, 421 Annotation: Subtype: Link Rect: 370, 387, 407, 398 Annotation: Subtype: Link Rect: 413, 387, 449, 398 Annotation: Subtype: Link Rect: 412, 364, 449, 375 Annotation: Subtype: Link Rect: 347, 341, 384, 352 Annotation: Subtype: Link Rect: 485, 318, 523, 329 Annotation: Subtype: Link Rect: 182, 295, 219, 306 Annotation: Subtype: Link Rect: 172, 273, 210, 283 Annotation: Subtype: Link Rect: 183, 238, 220, 249 Annotation: Subtype: Link Rect: 227, 238, 263, 249 Checksum: db83c589 Type: CRC32 Checksum: 8fdcf574c3ec83091f98dca2aa6637ef Type: MD5 Checksum: 3a95a1c34afc57600b260daf1ff051e35bc5d765 Type: SHA-1