Erythropoietin preserves the endothelial differentiation potential of cardiac progenitor cells and attenuates heart failure during anti-cancer therapy

Transcription

1 Erythropoietin preserves the endothelial differentiation potential of cardiac progenitor cells and attenuates heart failure during anti-cancer therapy Melanie Hoch Cardiology & Angiology MHH, Hannover

2 Situations where hearts are prone to develop failure include treatment concepts for human malignancies. The anthracyclin doxorubicin (DOX), has been used effectively to treat a broad range of cancers. Its clinical usage and efficacy, however, is limited by side effects, especially cardiomyopathy and heart failure (Ferreira et al., 28). Blocking STAT3 signaling by antisense, RNA interference (RNAi), peptides, and small molecular inhibitors appears successful in suppression of tumor cell growth and apoptosis making STAT3 an attractive molecular target for the development of novel cancer therapeutics (Alas and Bonavida, 23; Deng et al., 27). In turn, observations from our and other labs clearly show that downregulation of STAT3 pre-disposes the heart to failure (Hilfiker-Kleiner et al., 24; Hilfiker-Kleiner et al., 27; Jacoby et al., 23).

3 Early and late-onset of cardiomyopathy after cardiotoxic treatments Early-onset cardiotoxicity: Onset within one year after completion of treatment with a chronic dilated cardiomyopathy. Late-onset cardiotoxicity: Onset of progressive left ventricular dysfunction leading to irreversible congestive heart failure up to 15 years after treatment. Late-onset heart failure seems often triggered by events such as exercise, pregnancy, and acute viral infection. Frequency of cardiotoxic effects in survivors of childhood cancer: up to 57%.

4 Hypothesis for late-onset cardiomyopathy: Chemotherapy impairs the endogenous cardiac regeneration potential of the heart by altering the cardiac microenvironment. cardiomyocyte cardiac function cardiac remodeling paracrine factors cardiac vascularization cardiac microenvironment

5 count count Isolation of cardiac Sca-1 + progenitor cells (CPC) by the MACS system Male mice (age 3 to 4 months) Sca-1 + cells before selection post selection > 92% Sca-1 + FACS Characterization Marker Expression Marker Expression ckit 1% CD13 45% CD133 4% CD73 6% CD16 % CD29 13% FLK-1 % CD44 17% CD144 % CD9 28% CD34 % CD15 25% CD45 % CD31 3% No expression of hematopoietic or endothelial progenitors. Expression of mesenchymal markers CD13, CD73, CD29, CD44, and CD9 Sca-1-FITC RT-PCR Foxa2 Brachyury Oct4 Nanog Sox2 GAPDH Sca-1-FITC ES Flow CPC H 2 O CPC, but not the non-cpc cell fraction express nanog and brachyury. Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

6 Subpopulations of Sca-1 + CPC differentiate into vascular endothelial cells, pericytes/fibroblasts and adipocytes Endothelial cells Pericytes/Fibroblasts VE-Cadherin Cre tg/+ ; ROSA flox/+ CPC Cre+ 4W CPC Cre+ Flow Cre+ Sca-1/vimentin Sca-1/sm-α-actin Sca-1/NG2 VE-Cad GAPDH enos/dapi CPC fresh CPC 4W LV VE-Cadherin/DAPI (%) VE-Cadherin mrna VEGFR3/DAPI CPC fresh CPC 4W Adipocytes Oil Red IB4/DAPI FLK-1/DAPI VEGFR3/IB4/DAPI Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

7 count count CCR2 and R double-positive WT-CPC exert a high endothelial differentiation potential FACS Characterization 29% R + 28% CCR2 + Sca-1 + IB4/DAPI Sca-1 +/ R + /CCR2 + IB4/DAPI R-APC CCR2-APC Freshly isolated WT-CPC CCR2/DAPI Sca-1/DAPI R/DAPI Overlay Branching Points (%) 25 2 Sca-1 + Sca-1 + / R + /CCR2 + Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

8 Clonal analysis confirms that CCR2 and R double-positive CPC differentiate into vascular endothelial cells Clonal analysis Clone Sca-1 Nanog E-Cadherin VE-cadherin Endothelial Differentiation C C18 Nd C C C C27 Nd C Clonal analysis C25 in matrigel VE-Cadherin DAPI RT-PCR C25 C26 VE-Cadherin/DAPI VEGFR3 CCR2 R GAPDH Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

9 CPC express the CCR2 ligand but not R CCR2 CPC HEK 293 /DAPI /DAPI CPC Liver IgG/DAPI RT-PCR K CPC IgG RT-PCR L CPC GAPDH CPC bind in the cardiac microenvironment /Sca-1/DAPI /Sca-1/DAPI GAPDH Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

10 Activation of CCR2 is required for endothelial differentiation of CPC R CCR2 Impaired endothelial differentiation of CCR2-KO-CPC or WT-CPC with CCR2-Inhibitor control RS CCR2-KO (%) 15 Branching Points 1 5 n=3 independent isolations (each isolation: 1 mice) WT-CPC WT-CPC, RS CCR2-KO-CPC p<.5, p<.5 Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

11 Hypothesis for late-onset cardiomyopathy: Blockade of cardiac STAT3 impairs the endogenous cardiac regeneration by altering the cardiac microenvironment. cardiomyocyte STAT3 paracrine factors cardiac function cardiac remodeling cardiac vascularization cardiac microenvironment

12 Cardiomyocyte-specific deficiency for STAT3 pre-disposes to heart failure in response to age-, pregnancy-, I/R- and doxorubicin-mediated stress Hilfiker-Kleiner, D et al., Circ Res 24 Hilfiker-Kleiner, D et al., Cell 27 Hilfiker-Kleiner, D et al., Circ Res 24 Negoro et al., CVR 2 Kunisada et al., PNAS 2 cardiomyocyte STAT3 paracrine factors cardiac function cardiac vascularization endothelial differentiation cardiac microenvironment human heart failure STAT3 ptyrstat3/stat3 Reduced expression and activation of STAT3 in the failing human heart (Podewski et al. Circ. 23)

13 Impaired endothelial differentiation of CPC from mice with a cardiomyocyte-specific STAT3 deletion (CKO) compared to CPC from WT hearts WT CKO Sca-1 + cells STAT3 WT-CPC Equal STAT3 protein levels in WT- and CKO-CPC WT CPC CKO STAT3 STAT3 CKO-CPC Actin Sca-1 + CPC from CKO mice displayed impaired endothelial differentiation in vitro Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

14 CCR2 Cy3 FL2-H: CCR2 Cy3 CCR2 Cy3 FL2-H: CCR2 Cy3 % of Max % of Max The impaired endothelial net formation of CKO-CPC was associated with a reduced CCR2 expression R CCR2 FACS Analysis WT- vs. CKO-CPC WT CCR2 CKO CCR R 1 8 CCR FL1-H: SCA1 FITC Sca-1 FITC FL1-H: SCA1 FITC Sca-1 FITC R Cy CCR2 Cy3 WT-CPC CKO-CPC Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

15 and with increased generation of antagonistic achieved by up-regulated MMP-12 expression MMP-12 Micro array analysis CKO-CPC Fold change compared to WT-CPC Cleavage Assay WT CKO r cl PF Addition of and MMP-12 inhibitor improves endothelial differentiation in CKO-CPC R. Dean et al. 28, Blood Impaired endothelial differentiation of MMP-12 Branching Points (%) Branching Points CKO-CPC CKO-CPC, CKO-CPC, +PF control rmmp-12 (%) WT-CPC WT-CPC, MMP-12 Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

16 expression was markedly reduced in CKO hearts. Is there a connection between low cardiac, blocked CCR2 signaling on CPCs and their impaired angiongenic potential? Impaired endothelial differentiation CKO-CPC Altered /CCR2/MMP-12 system in CKO-CPC MMP-12 paracrine factors? Cardiomyocyte STAT3- deficiency STAT3 cl. Micro array analysis of CKO hearts /Sca-1/DAPI Fold change compared to WT hearts Is a paracrine factor that acts in the cardiac microenvironment?

17 expression in normoxic hearts depends in part on STAT3 IHC of murine LV sections WT CKO Epo-TAg h Western Blot WT CKO EpoTAg h /α-actinin /α-actinin /α-actinin Actin IgG Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

18 Expression in cultivated cardiomyocytes depends on STAT3 IHC of NRCM α-actinin//dapi Western Blot NRCM C KO STAT3 Actin C: Control KO: STAT3-Knockdown Methylcellulose-Assay (CFU) RT-PCR Methylcellulose-Assay (CFU) neg. control pos. control NRCM scramble scramble sirna kidney NRCM supernatant heat-inactivated NRCM supernatant GAPDH sirna Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

19 R MMP-12 CCR2 cl. r to CKO-CPC cultures improved their endothelial differentiation WT-CPC control r CKO-CPC control r CKO-CPC control r qrt-pcr after 4 weeks MMP-12 (%) 2 (%) CKO-CPC CKO-CPC, repo (%) Branching Points WT-CPC WT-CPC, repo ## Sprouting (%) CKO-CPC CKO-CPC, repo (%) VE-Cadherin mrna Luciferase Activity of the promoter (%) n=3; p<.5 vs. CKO, p<.1 vs. WT; ## p<.5 vs. CKO Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211 control r TNF

20 r can not improve endothelial differentiation of CCR2-KO-CPC or of WT-CPC with blocked CCR2 suggesting that induces endothelial differentiation in CPCs via CCR2 signaling. R MMP-12 CCR2 cl. WT-CPC with CCR2-inhibitor CCR2-KO-CPC WT-CPC IB4/DAPI WT-CPC, RS IB4/DAPI (%) 12 Branching Points n.s. (%) 12 Branching Points WT-CPC, RS-12895, r IB4/DAPI WT-CPC WT-CPC, RS WT-CPC, RS r n.s. WT-CPC CCR2-KO CPC CCR2-KO CPC,r n=3; p<.5 vs. WT, p<.1 vs. WT Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

21 Reconstitution of by treatment with CERA Has the reduction of myocardial levels an effect on the myocardial vascularisation? Cardiomyocyte CPC R STAT3 CCR2 CPC Cardiac Vasculogenesis endothelial differentiation

22 Treatment of CKO mice with the derivative CERA restored endothelial differentiation of CKO-CPC R MMP-12 CCR2 cl. Low-dose CERA (Continunous Erythropoiesis Receptor Activator) treatment over 3 weeks in vivo in a non-hematocrit influencing dosage (3 g/kgbw/week) 3 weeks in vivo treatment followed by 4 weeks of in vitro cultivation WT control CKO control qrt-pcr of freshly isolated CPC after 3 week in vivo treatment (%)15 MMP-12 (%) CPC WT CERA CKO CKO CERA 5 1 CKO-CPC, NaCl CKO-CPC, CERA n=3; p<.5 vs. WT-CPC Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

23 CERA improved capillary density and cardiac function without altering inflammatory status in CKO mice Capillary density (IHC WGA+IB4) CKO control CKO CERA CD45 staining CKO control CKO CERA Capillaries/Cardiomyocyte (%) CKO, NaCl CKO, CERA CD45 positive infiltrates (arb. unit) CKO, NaCl CKO, CERA Fractional Shortening (FS) n=5-8 animals; p<.5, p<.5 FS(%) basal NaCl CERA WT CKO Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

24 Hypothesis : DOX impairs the endogenous cardiac regeneration by altering the cardiac microenvironment in a similar way as KO of STAT3 since it is known that DOX down-regulates STAT3 in the heart (Kunisada et al. PNAS 2). DOX C,1,5 STAT3 WT DOX Actin (%) 12 protein Actin 8 4 We could confirm down-regulation of STAT3 by DOX and observed that DOX reduced also expression in cardiomyocytes in vitro and in vivo (Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211)

25 Treatment concept: Repetitive high dose of DOX (6 mg/kg/week for 3 weeks) induces high mortality associated with heart failure and reduced cardiac capillary density Survival curve Fractional Shortening (FS) Capillary density WT/NaCl, n=16 DOX/NaC, n=18 (%) WT NaCl DOX/NaCl (%) WT/NaCl DOX/NaCl days n=6 animals; p<.5, p<.5 Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

26 DOX treatment impairs endothelial differentiation of CPC which is associated with a marked down-regulation of cardiac expression R MMP-12 CCR2 DOX 3 weeks in vivo treatment followed by 4 weeks of in vitro cultivation cl. WT-mice/NaCl IB4/DAPI DOX-mice IB4/DAPI qrt-pcr of cultured CPC after 3 week in vivo treatment PCR (%) WT-CPC/NaCl (%) PCR MMP-12 DOX-CPC Branching points PCR VE-Cadherin (%) 2 (%) WT-CPC/NaCl DOX-CPC n=3 independent isolations (each isolation: 1 mice) p<.5, p<.5 Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

27 Co-treatment with the derivative CERA improved endothelial differentiation and restored expression in CPC from DOX treated mice 3 weeks in vivo treatment followed by 4 weeks of in vitro cultivation qrt-pcr of cultured CPC after 3 week in vivo treatment WT-mice/NaCl DOX-mice/NaCl DOX-mice/CERA IB4/DAPI IB4/DAPI IB4/DAPI (%)15 PCR (%) 15 PCR MMP Branching points (%) PCR VE-Cadherin (%) WT-CPC/NaCl DOX-CPC/NaCl DOX/CERA WT-CPC/NaCl DOX-CPC/NaCl DOX/CERA n=3 independent isolations (each isolation: 1 mice) p<.5, p<.5 Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

28 and it prevented decrease in cardiac capillary density and function and improved survival in DOX treated mice Survival curve Fractional Shortening (FS) Capillary density WT/NaCl, n=16 DOX/NaC, n=18 DOX/CERA, n=17 ## (%) WGA/IB4/DAPI WT NaCl DOX/NaCl DOX/CERA (%) WT/NaCl DOX/NaCl DOX/CERA n=6 animals; p<.5, p<.5 Hoch,..., Hilfiker-Kleiner, Cell Stem Cell 211

29 Summary The endothelial regeneration potential of CPCs is altered in hearts exposed to anti-tumor therapy regimes involving DOX or reduction in STAT3. Alterations in the cardiac microenvironment are responsible for modifications of the autocrine /CCR2 system that is needed for endothelial differentiation of CPC. is a component of the cardiac microenvironment that is disturbed by DOX and cardiomyocyte STAT3 reduction. treatment improves endothelial differentiation of CPCs via activation of CCR2, preserves the cardiac vasculature and prevents heart failure in mice harboring a cardiac STAT3 deficiency or obtaining DOX treatment.

30 Conclusion Schneider, editiorial, Cell Stem Cell 211 Diseased hearts display an altered cardiac micorenvironment that affects the regeneration potential of progenitor cells. Exogenous (hematocrit-inactive) may help to restore the cardiac microenvironment thereby improving cardiac regeneration during anti-tumor drug therapy and may be even in a more general way to improve regenerative strategies of the heart involving cell therapeutic approaches.

31 Acknowledgements Hannover Medical School Molecular Cardiology D. Hilfiker-Kleiner B. Stapel P. Fischer A. Haghikia S. Erschow Molecular Biology K. Schuster-Gossler A. Gossler University of Louvain Medical School Experimental and Clinical Research Institute B. Sekkali J.-L. Balligand Haematology, Haemostasis, Oncology and Stem cell transplantation M. Scherr M. Eder University Paris F. Favret MPI Bad Nauheim T. Braun