d8577af8378beaf01519f57cf2354e47 pmc_34359858.pdf fada69f1f1bae790d20f994d6a24c1cab8c97dcb pmc_34359858.pdf b66edaf768fe64c3f0f3ed9d7027e7114ab882b8354482f11956809664312b2e pmc_34359858.pdf Title: Circulating Cell Biomarkers in Pulmonary Arterial Hypertension: Relationship with Clinical Heterogeneity and Therapeutic Response Subject: Background: Endothelial dysfunction is central to PAH. In this study, we simultaneously analysed circulating levels of endothelial microvesicles (EMVs) and progenitor cells (PCs) in PAH and in controls, as biomarkers of pulmonary endothelial integrity and evaluated differences among PAH subtypes and as a response to treatment. Methods: Forty-seven controls and 144 patients with PAH (52 idiopathic, 9 heritable, 31 associated with systemic sclerosis, 15 associated with other connective tissue diseases, 20 associated with HIV and 17 associated with portal hypertension) were evaluated. Forty-four patients with scleroderma and 22 with HIV infection, but without PAH, were also studied. Circulating levels of EMVs, total (CD31+CD42b-) and activated (CD31+CD42b-CD62E+), as well as circulating PCs (CD34+CD133+CD45low) were measured by flow cytometry and the EMVs/PCs ratio was computed. In treatment-naïve patients, measurements were repeated after 3 months of PAH therapy. Results: Patients with PAH showed higher numbers of EMVs and a lower percentage of PCs, compared with healthy controls. The EMV/PC ratio was increased in PAH patients, and in patients with SSc or HIV without PAH. After starting PAH therapy, individual changes in EMVs and PCs were variable, without significant differences being observed as a group. Conclusion: PAH patients present disturbed vascular homeostasis, reflected in changes in circulating EMV and PC levels, which are not restored with PAH targeted therapy. Combined measurement of circulating EMVs and PCs could be foreseen as a potential biomarker of endothelial dysfunction in PAH. Keywords: pulmonary arterial hypertension; endothelial dysfunction; biomarkers; progenitor cells; endothelial extracellular vesicles; PAH-specific treatment Author: Olga Tura-Ceide, Isabel Blanco, Jéssica Garcia-Lucio, Roberto del Pozo, Agustín Roberto García, Elisabet Ferrer, Isabel Crespo, Diego A. Rodríguez-Chiaradia, Carmen Pilar Simeon-Aznar, Manuel López-Meseguer, Clara Martín-Ontiyuelo, Víctor I. Peinado and Joan Albert Barberà Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Jul 6 04:35:53 2021 CEST ModDate: Tue Jul 6 04:40:29 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1288822 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- KORLVE+VnURWPalladioL Type 1 Custom yes yes no 10 0 TQCAXE+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 IDRBQJ+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 XGMDJV+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 NTJMOU+CMSY10 Type 1 Builtin yes yes no 28 0 ZSBPMM+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 PFKOHU+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 40 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 62 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 66 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 70 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 82 0 WABLSD+SFRM1000 Type 1 Custom yes yes no 95 0 FHBIAZ+EURB7 Type 1 Builtin yes yes no 106 0 AJJIAH+Arial,Italic TrueType WinAnsi yes yes no 117 0 AJJFJN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 120 0 AJJFJO+PalatinoLinotype CID TrueType Identity-H yes yes yes 123 0 AJJFJP+PalatinoLinotype TrueType WinAnsi yes yes no 129 0 AJJFKB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 132 0 AJJHPD+Calibri CID TrueType Identity-H yes yes yes 135 0 AJJHPE+Arial,Bold CID TrueType Identity-H yes yes yes 141 0 AJJHPF+Arial CID TrueType Identity-H yes yes yes 147 0 AJJIAH+Arial,Italic TrueType WinAnsi yes yes no 163 0 AJJFJN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 166 0 AJJFJP+PalatinoLinotype TrueType WinAnsi yes yes no 169 0 AJJFKB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 172 0 AJJHPD+Calibri CID TrueType Identity-H yes yes yes 175 0 AJJHPE+Arial,Bold CID TrueType Identity-H yes yes yes 181 0 AJJHPF+Arial CID TrueType Identity-H yes yes yes 187 0 AJJIAH+Arial,Italic TrueType WinAnsi yes yes no 203 0 AJJFJN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 206 0 AJJFJO+PalatinoLinotype CID TrueType Identity-H yes yes yes 209 0 AJJFJP+PalatinoLinotype TrueType WinAnsi yes yes no 215 0 AJJFKB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 218 0 AJJHPD+Calibri CID TrueType Identity-H yes yes yes 221 0 AJJHPE+Arial,Bold CID TrueType Identity-H yes yes yes 227 0 AJJHPF+Arial CID TrueType Identity-H yes yes yes 233 0 AJJFJN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 249 0 AJJFJP+PalatinoLinotype TrueType WinAnsi yes yes no 252 0 AJJFKB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 255 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:08:15 CEST RepresentationInformation: pmc_34359858.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:14:45 CEST Size: 1288822 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 490 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Study Population Destination: subsection.2.1 Item: Title: Measurements Destination: subsection.2.2 Item: Title: Assessment of Circulating Endothelial Microvesicles (EMVs) Destination: subsection.2.3 Item: Title: Evaluation of Circulating Progenitor Cells (PCs) Destination: subsection.2.4 Item: Title: Statistical Analysis Destination: subsection.2.5 Item: Title: Results Destination: section.3 Children: Item: Title: Population Characteristics Destination: subsection.3.1 Item: Title: Circulating Endothelial Microvesicles (EMVs) Destination: subsection.3.2 Item: Title: Circulating Progenitor Cells (PCs) Destination: subsection.3.3 Item: Title: EMVs/PCs Ratio Destination: subsection.3.4 Item: Title: Changes Induced by PAH Treatment Destination: subsection.3.5 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: section.4 Info: Title: Circulating Cell Biomarkers in Pulmonary Arterial Hypertension: Relationship with Clinical Heterogeneity and Therapeutic Response Author: Olga Tura-Ceide, Isabel Blanco, Jéssica Garcia-Lucio, Roberto del Pozo, Agustín Roberto García, Elisabet Ferrer, Isabel Crespo, Diego A. Rodríguez-Chiaradia, Carmen Pilar Simeon-Aznar, Manuel López-Meseguer, Clara Martín-Ontiyuelo, Víctor I. Peinado and Joan Albert Barberà Subject: Background: Endothelial dysfunction is central to PAH. In this study, we simultaneously analysed circulating levels of endothelial microvesicles (EMVs) and progenitor cells (PCs) in PAH and in controls, as biomarkers of pulmonary endothelial integrity and evaluated differences among PAH subtypes and as a response to treatment. Methods: Forty-seven controls and 144 patients with PAH (52 idiopathic, 9 heritable, 31 associated with systemic sclerosis, 15 associated with other connective tissue diseases, 20 associated with HIV and 17 associated with portal hypertension) were evaluated. Forty-four patients with scleroderma and 22 with HIV infection, but without PAH, were also studied. Circulating levels of EMVs, total (CD31+CD42b-) and activated (CD31+CD42b-CD62E+), as well as circulating PCs (CD34+CD133+CD45low) were measured by flow cytometry and the EMVs/PCs ratio was computed. In treatment-naïve patients, measurements were repeated after 3 months of PAH therapy. Results: Patients with PAH showed higher numbers of EMVs and a lower percentage of PCs, compared with healthy controls. The EMV/PC ratio was increased in PAH patients, and in patients with SSc or HIV without PAH. After starting PAH therapy, individual changes in EMVs and PCs were variable, without significant differences being observed as a group. Conclusion: PAH patients present disturbed vascular homeostasis, reflected in changes in circulating EMV and PC levels, which are not restored with PAH targeted therapy. Combined measurement of circulating EMVs and PCs could be foreseen as a potential biomarker of endothelial dysfunction in PAH. Keywords: pulmonary arterial hypertension; endothelial dysfunction; biomarkers; progenitor cells; endothelial extracellular vesicles; PAH-specific treatment Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Jul 06 04:35:53 CEST 2021 ModDate: Tue Jul 06 04:40:29 CEST 2021 ID: 0x60baef4d85a06a18e7073ffeeeec37e4, 0x60baef4d85a06a18e7073ffeeeec37e4 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: ZSBPMM+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: ZSBPMM+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: IDRBQJ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: IDRBQJ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XGMDJV+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: XGMDJV+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: KORLVE+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: KORLVE+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: FHBIAZ+EURB7 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: FHBIAZ+EURB7 Flags: Symbolic FontBBox: -1, -247, 1316, 723 FontFile: true Font: BaseFont: NTJMOU+CMSY10 FontSubset: true FirstChar: 0 LastChar: 21 FontDescriptor: FontName: NTJMOU+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: TQCAXE+URWPalladioL-Ital FontSubset: true FirstChar: 40 LastChar: 121 FontDescriptor: FontName: TQCAXE+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WABLSD+SFRM1000 FontSubset: true FirstChar: 180 LastChar: 180 FontDescriptor: FontName: WABLSD+SFRM1000 Flags: Symbolic FontBBox: -189, -321, 1456, 937 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 122, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 136, 641, 147, 654 Annotation: Subtype: Link Rect: 107, 628, 118, 642 Annotation: Subtype: Link Rect: 131, 346, 154, 355 Annotation: Subtype: Link Rect: 35, 334, 152, 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: 104, 17, 244, 28 Annotation: Subtype: Link Rect: 419, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 470, 87, 489 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 248, 737, 255, 746 ActionDest: 266 Annotation: Subtype: Link Rect: 256, 737, 263, 746 ActionDest: 266 Annotation: Subtype: Link Rect: 325, 711, 332, 721 ActionDest: 266 Annotation: Subtype: Link Rect: 438, 699, 445, 708 ActionDest: 266 Annotation: Subtype: Link Rect: 363, 636, 370, 645 ActionDest: 269 Annotation: Subtype: Link Rect: 401, 611, 408, 620 ActionDest: 269 Annotation: Subtype: Link Rect: 410, 573, 417, 582 ActionDest: 269 Annotation: Subtype: Link Rect: 522, 511, 529, 520 ActionDest: 269 Annotation: Subtype: Link Rect: 502, 485, 509, 495 ActionDest: 269 Annotation: Subtype: Link Rect: 288, 435, 295, 444 ActionDest: 269 Annotation: Subtype: Link Rect: 214, 398, 226, 407 ActionDest: 269 Annotation: Subtype: Link Rect: 241, 385, 252, 394 ActionDest: 269 Annotation: Subtype: Link Rect: 213, 360, 225, 369 ActionDest: 269 Annotation: Subtype: Link Rect: 505, 360, 517, 369 ActionDest: 269 Annotation: Subtype: Link Rect: 285, 335, 297, 344 ActionDest: 269 Annotation: Subtype: Link Rect: 376, 297, 383, 306 ActionDest: 269 Annotation: Subtype: Link Rect: 384, 297, 396, 306 ActionDest: 269 Annotation: Subtype: Link Rect: 396, 297, 408, 306 ActionDest: 269 Annotation: Subtype: Link Rect: 321, 272, 333, 281 ActionDest: 269 Annotation: Subtype: Link Rect: 511, 247, 523, 256 ActionDest: 269 Annotation: Subtype: Link Rect: 289, 234, 301, 244 ActionDest: 269 Annotation: Subtype: Link Rect: 304, 234, 316, 244 ActionDest: 269 Annotation: Subtype: Link Rect: 224, 159, 236, 168 ActionDest: 269 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 531, 607, 538, 619 ActionDest: 86 Annotation: Subtype: Link Rect: 217, 143, 224, 152 ActionDest: 269 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 290, 669, 297, 678 ActionDest: 266 Annotation: Subtype: Link Rect: 366, 657, 373, 666 ActionDest: 269 Annotation: Subtype: Link Rect: 496, 493, 508, 502 ActionDest: 269 Annotation: Subtype: Link Rect: 424, 242, 436, 251 ActionDest: 269 Annotation: Subtype: Link Rect: 544, 91, 556, 100 ActionDest: 269 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 516, 634, 528, 643 ActionDest: 269 Annotation: Subtype: Link Rect: 507, 584, 519, 593 ActionDest: 269 Annotation: Subtype: Link Rect: 235, 379, 241, 391 ActionDest: 86 Annotation: Subtype: Link Rect: 483, 329, 490, 341 ActionDest: 86 Annotation: Subtype: Link Rect: 522, 329, 529, 341 ActionDest: 100 Annotation: Subtype: Link Rect: 529, 304, 536, 316 ActionDest: 100 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 273, 432, 280, 444 ActionDest: 103 Annotation: Subtype: Link Rect: 334, 432, 341, 444 ActionDest: 153 Annotation: Subtype: Link Rect: 443, 407, 450, 419 ActionDest: 103 Annotation: Subtype: Link Rect: 325, 369, 332, 381 ActionDest: 153 Annotation: Subtype: Link Rect: 522, 344, 528, 356 ActionDest: 103 Annotation: Subtype: Link Rect: 195, 306, 202, 318 ActionDest: 153 Annotation: Subtype: Link Rect: 353, 294, 360, 306 ActionDest: 103 Annotation: Subtype: Link Rect: 413, 294, 420, 306 ActionDest: 153 Annotation: Subtype: Link Rect: 455, 243, 462, 257 ActionDest: 153 Annotation: Subtype: Link Rect: 517, 243, 524, 257 ActionDest: 153 Annotation: Subtype: Link Rect: 525, 218, 532, 230 ActionDest: 153 Annotation: Subtype: Link Rect: 541, 193, 548, 205 ActionDest: 153 Annotation: Subtype: Link Rect: 322, 156, 329, 168 ActionDest: 153 Annotation: Subtype: Link Rect: 318, 80, 325, 92 ActionDest: 193 Annotation: Subtype: Link Rect: 437, 67, 444, 79 ActionDest: 193 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 410, 720, 417, 732 ActionDest: 193 Page: Sequence: 8 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 215, 682, 222, 694 ActionDest: 239 Annotation: Subtype: Link Rect: 258, 233, 270, 243 ActionDest: 272 Annotation: Subtype: Link Rect: 273, 233, 285, 243 ActionDest: 272 Annotation: Subtype: Link Rect: 326, 221, 333, 230 ActionDest: 269 Annotation: Subtype: Link Rect: 334, 221, 346, 230 ActionDest: 269 Annotation: Subtype: Link Rect: 395, 208, 407, 217 ActionDest: 269 Annotation: Subtype: Link Rect: 407, 208, 419, 217 ActionDest: 272 Annotation: Subtype: Link Rect: 231, 171, 243, 180 ActionDest: 272 Annotation: Subtype: Link Rect: 239, 133, 251, 142 ActionDest: 269 Annotation: Subtype: Link Rect: 252, 133, 264, 142 ActionDest: 272 Annotation: Subtype: Link Rect: 433, 120, 445, 130 ActionDest: 272 Annotation: Subtype: Link Rect: 448, 120, 460, 130 ActionDest: 272 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 414, 609, 426, 618 ActionDest: 272 Annotation: Subtype: Link Rect: 399, 584, 411, 593 ActionDest: 272 Annotation: Subtype: Link Rect: 453, 559, 465, 568 ActionDest: 272 Annotation: Subtype: Link Rect: 432, 496, 444, 505 ActionDest: 272 Annotation: Subtype: Link Rect: 528, 471, 540, 480 ActionDest: 272 Annotation: Subtype: Link Rect: 543, 471, 555, 480 ActionDest: 272 Annotation: Subtype: Link Rect: 329, 459, 341, 468 ActionDest: 272 Annotation: Subtype: Link Rect: 341, 459, 353, 468 ActionDest: 272 Annotation: Subtype: Link Rect: 466, 459, 478, 468 ActionDest: 272 Annotation: Subtype: Link Rect: 290, 421, 302, 430 ActionDest: 272 Annotation: Subtype: Link Rect: 446, 320, 458, 330 ActionDest: 272 Annotation: Subtype: Link Rect: 268, 270, 279, 279 ActionDest: 272 Annotation: Subtype: Link Rect: 321, 245, 333, 254 ActionDest: 272 Annotation: Subtype: Link Rect: 455, 182, 467, 192 ActionDest: 272 Annotation: Subtype: Link Rect: 344, 95, 356, 104 ActionDest: 269 Annotation: Subtype: Link Rect: 356, 95, 368, 104 ActionDest: 269 Annotation: Subtype: Link Rect: 196, 70, 208, 79 ActionDest: 269 Annotation: Subtype: Link Rect: 391, 69, 402, 79 ActionDest: 269 Annotation: Subtype: Link Rect: 406, 69, 418, 79 ActionDest: 269 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 523, 710, 535, 719 ActionDest: 269 Annotation: Subtype: Link Rect: 424, 597, 436, 606 ActionDest: 272 Annotation: Subtype: Link Rect: 431, 572, 443, 581 ActionDest: 272 Annotation: Subtype: Link Rect: 226, 484, 238, 493 ActionDest: 269 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 424, 633, 560, 644 Annotation: Subtype: Link Rect: 166, 621, 271, 632 Annotation: Subtype: Link Rect: 125, 141, 310, 152 Annotation: Subtype: Link Rect: 422, 141, 460, 152 Annotation: Subtype: Link Rect: 87, 108, 125, 118 Annotation: Subtype: Link Rect: 131, 108, 167, 118 Annotation: Subtype: Link Rect: 255, 72, 496, 83 Annotation: Subtype: Link Rect: 108, 61, 145, 72 Annotation: Subtype: Link Rect: 152, 61, 188, 72 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 125, 709, 310, 720 Annotation: Subtype: Link Rect: 425, 709, 463, 720 Annotation: Subtype: Link Rect: 378, 686, 561, 697 Annotation: Subtype: Link Rect: 171, 675, 208, 686 Annotation: Subtype: Link Rect: 215, 675, 251, 686 Annotation: Subtype: Link Rect: 56, 640, 241, 651 Annotation: Subtype: Link Rect: 356, 640, 394, 651 Annotation: Subtype: Link Rect: 400, 640, 436, 651 Annotation: Subtype: Link Rect: 59, 607, 97, 617 Annotation: Subtype: Link Rect: 103, 607, 139, 617 Annotation: Subtype: Link Rect: 86, 571, 271, 582 Annotation: Subtype: Link Rect: 386, 571, 424, 582 Annotation: Subtype: Link Rect: 430, 571, 466, 582 Annotation: Subtype: Link Rect: 338, 548, 521, 559 Annotation: Subtype: Link Rect: 132, 537, 169, 548 Annotation: Subtype: Link Rect: 125, 491, 310, 502 Annotation: Subtype: Link Rect: 425, 491, 463, 502 Annotation: Subtype: Link Rect: 379, 456, 561, 467 Annotation: Subtype: Link Rect: 171, 445, 208, 456 Annotation: Subtype: Link Rect: 56, 410, 241, 421 Annotation: Subtype: Link Rect: 356, 410, 394, 421 Annotation: Subtype: Link Rect: 400, 410, 436, 421 Annotation: Subtype: Link Rect: 377, 387, 561, 398 Annotation: Subtype: Link Rect: 171, 376, 208, 387 Annotation: Subtype: Link Rect: 188, 341, 373, 352 Annotation: Subtype: Link Rect: 489, 341, 526, 352 Annotation: Subtype: Link Rect: 109, 273, 146, 283 Annotation: Subtype: Link Rect: 453, 249, 560, 260 Annotation: Subtype: Link Rect: 56, 238, 135, 249 Annotation: Subtype: Link Rect: 250, 238, 288, 249 Annotation: Subtype: Link Rect: 56, 203, 241, 214 Annotation: Subtype: Link Rect: 356, 203, 394, 214 Annotation: Subtype: Link Rect: 400, 203, 436, 214 Annotation: Subtype: Link Rect: 536, 180, 560, 191 Annotation: Subtype: Link Rect: 56, 169, 217, 180 Annotation: Subtype: Link Rect: 179, 134, 363, 145 Annotation: Subtype: Link Rect: 478, 134, 515, 145 Annotation: Subtype: Link Rect: 521, 134, 557, 145 Annotation: Subtype: Link Rect: 410, 111, 560, 122 Annotation: Subtype: Link Rect: 56, 100, 90, 111 Annotation: Subtype: Link Rect: 205, 100, 243, 111 Annotation: Subtype: Link Rect: 56, 65, 241, 76 Annotation: Subtype: Link Rect: 356, 65, 394, 76 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 536, 721, 560, 732 Annotation: Subtype: Link Rect: 56, 709, 217, 720 Annotation: Subtype: Link Rect: 333, 709, 370, 720 Annotation: Subtype: Link Rect: 56, 686, 244, 697 Annotation: Subtype: Link Rect: 359, 686, 397, 697 Annotation: Subtype: Link Rect: 478, 663, 560, 674 Annotation: Subtype: Link Rect: 56, 652, 156, 663 Annotation: Subtype: Link Rect: 272, 652, 309, 663 Annotation: Subtype: Link Rect: 316, 652, 352, 663 Annotation: Subtype: Link Rect: 256, 617, 445, 628 Annotation: Subtype: Link Rect: 59, 607, 97, 617 Annotation: Subtype: Link Rect: 222, 571, 405, 582 Annotation: Subtype: Link Rect: 520, 571, 557, 582 Annotation: Subtype: Link Rect: 59, 561, 95, 571 Annotation: Subtype: Link Rect: 418, 537, 560, 548 Annotation: Subtype: Link Rect: 56, 525, 99, 536 Annotation: Subtype: Link Rect: 215, 525, 252, 536 Annotation: Subtype: Link Rect: 251, 502, 560, 513 Annotation: Subtype: Link Rect: 171, 491, 208, 502 Annotation: Subtype: Link Rect: 56, 456, 241, 467 Annotation: Subtype: Link Rect: 356, 456, 394, 467 Annotation: Subtype: Link Rect: 308, 422, 560, 433 Annotation: Subtype: Link Rect: 171, 410, 208, 421 Annotation: Subtype: Link Rect: 269, 376, 451, 387 Annotation: Subtype: Link Rect: 59, 365, 97, 375 Annotation: Subtype: Link Rect: 103, 365, 139, 375 Annotation: Subtype: Link Rect: 191, 330, 451, 341 Annotation: Subtype: Link Rect: 59, 319, 97, 329 Annotation: Subtype: Link Rect: 103, 319, 139, 329 Annotation: Subtype: Link Rect: 162, 284, 347, 295 Annotation: Subtype: Link Rect: 462, 284, 500, 295 Annotation: Subtype: Link Rect: 392, 249, 560, 260 Annotation: Subtype: Link Rect: 56, 238, 72, 249 Annotation: Subtype: Link Rect: 188, 238, 225, 249 Annotation: Subtype: Link Rect: 267, 203, 450, 214 Annotation: Subtype: Link Rect: 59, 192, 97, 203 Annotation: Subtype: Link Rect: 103, 192, 139, 203 Annotation: Subtype: Link Rect: 56, 157, 250, 168 Annotation: Subtype: Link Rect: 363, 157, 400, 168 Annotation: Subtype: Link Rect: 311, 134, 497, 145 Annotation: Subtype: Link Rect: 108, 123, 145, 134 Annotation: Subtype: Link Rect: 536, 111, 560, 122 Annotation: Subtype: Link Rect: 56, 100, 217, 111 Annotation: Subtype: Link Rect: 333, 100, 370, 111 Checksum: 089fb2d4 Type: CRC32 Checksum: d8577af8378beaf01519f57cf2354e47 Type: MD5 Checksum: fada69f1f1bae790d20f994d6a24c1cab8c97dcb Type: SHA-1 Checksum: b66edaf768fe64c3f0f3ed9d7027e7114ab882b8354482f11956809664312b2e Type: SHA-256