Mesenchymal precursor cells modulate the effect of bacterial endotoxin on inflammatory gene expression in human cells of monocyte lineage

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Heart, Lung and Circulation 2008;17S:S4–S53

Poster Presentations

Methods: MI was produced in male Wistar rats by left coronary ligation. Rats were sacrificed to obtain the heart at 3, 6, 15, 30 and 45 days after operation. Heart were cut into four cross sections, fixed and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin (HE), Masson’s trichrome (MT), rabbit anti-CRBP1 antibody and mouse anti-␣-smooth muscle (SM) actin antibody. CRBP-1 and ␣-SM actin were also detected using Western blotting. Results: Seventy Wistar rats developed MI with infarct size ranging from 43.3% to 58.1% (mean 48.8 ± 3.6%). Heart sections of MI with HE and MT staining showed a remarkable myocyte necrosis, collagen disposition and ventricular remodeling. CRBP-1 expression was detected at both of the endocardial and epicardial region of infarction, where fibroblasts infiltrated with myocyte necrosis at 3 days and 6 days after operation. At 15 days, ␣-SM actin positive fibroblasts in the infarcted region expressed CRBP-1 and this expression decreased at 30 days after operation. Conclusions: We demonstrate that CRBP-1 is transiently and rapidly expressed by myofibroblast in rat model of MI. Our results suggest that CRBP-1 and possibly retinoic acid play an important role in ventricular remodelling after MI. doi:10.1016/j.hlc.2007.11.131 Analysis of cardiac troponin I phosphorylation and degradation in injured cardiomyocytes Chun-Xia Zhu a,∗ , Heng-Fang Wu b , Ji-Zheng Ma b , ShuShu Zhu b , Xiaang-Jian Chen b a Nanjing

Chest Hospital, China

b Institute of Cardiovascular Disease of First Affiliated Hospital

with Nanjing, China To investigate the pathogenesis of cardiomyopathies at molecular and cellular level and provide the potential molecular pathological mechanisms. Methods: Neonatal rat cardiomyocytes were exposed to isoproterenol (10–4 mol/L, 10–5 mol/L) or cultured in sugar-serum-oxygen free medium for 2 h, 4 h, 6 h, respectively as a cell injury model. The cTnI in cultured medium was determined by using ELISA. Western Blotting was employed to determine the phosphorylated or dephosphorylated cTnI, PKC (␤1, ␤2, ␥) and cTnI degradation expression in injury cell. Quantity One 4.5.2 Quantitation software (Bio-Rad, USA) were used to perform semiquantification. Results: cTnI of cultured medium in cardiomyoctyes exposed with ISO increased markedly compared with control (P < 0.01). cTnI increased significantly in cultured cardiomyoctyes with sugar-serum-oxygen free medium for 2 h, 4 h, 6 h, respectively (P < 0.01), cTnI of cultured medium in cardiomyoctyes exposed by ISO were significantly higher than in sugar-serum-oxygen free medium (P < 0.05). There were no dephosphorylated cTnI and PKC␤1, PKC␤2, PKC␥ expressed in any group. The 28 kDa cTnI degradation bands showed in groups except control group detected by 8I-7 and in groups except control

group or sugar-serum-oxygen free medium for 2 h group detected by MF4. Conclusions: These results indicate that on the one side, cTnI was degraded from the N-terminal, on the other side, the cTnI affinity with anti-cTnI antibody decrease via the high concentration ISO or long time sugar-serumoxygen free medium intervention. The pathway induced cTnI phosphorylation in vitro cultured cardiomyocytes exposed with ISO or sugar-serum-oxygen free medium do not through PKC␤1, PKC␤2 and PKC␥. doi:10.1016/j.hlc.2007.11.132 Mesenchymal precursor cells modulate the effect of bacterial endotoxin on inflammatory gene expression in human cells of monocyte lineage Hendrik Zimmet a,∗ , Andrew Kompa a , Bing Wang a , Silviu Itescu b , Henry Krum b a Monash

University, Melbourne, Australia Of Melbourne, Melbourne, Australia

b University

Background: Activated monocytes, and their descendant macrophages, play a pivotal role in the evolution of coronary atheromatous plaques and ventricular remodeling post-myocardial infarction. Mesenchymal precursor cells (MPCs) have immunomodulatory properties in preventing T cell activation via inhibition of differentiation and function of monocyte-derived dendritic cells. We investigated MPCs modulatory effect on THP-1 monocyte activation. Methods: THP-1 cells and MPCs were cultured separately in serum-free conditions for 24 hours. Then the two cell types were cocultured in 12 well plates with transwell inserts, in MPC:THP-1 ratios of 0:1, 1:10, 1:20, 1:50 and exposed to 0.5 g/ml lipopolysaccharide (LPS) or vehicle for 24 h. The 0.4 m insert pore size allowed cytokine flux between the two cell types but not direct contact. THP-1 cells were harvested after coculture, total RNA extracted and real-time PCR performed to assess gene expression of TNF and IL-1. Cell culture and PCR samples were performed in triplicate. RPL32 was used as the housekeeping gene. Results: There were no significant differences between THP-1 cell TNF or IL-1 gene expression at baseline across the four groups (p = 0.103). Baseline TNF gene expression (mean TNF/RPL32 ratio) was 0.1046, 0.1585, 0.1946 and 0.3168 in the MPC:THP-1 cell ratio groups of 0:1, 1:50, 1:20 and 1:10, respectively. Percentage increase in TNF gene expression following LPS stimulation were 169.02, 61.07, 19.84 and 45.23% in the MPC:THP-1 cell ratio groups of 0:1, 1:50, 1:20 and 1:10, respectively. Baseline IL-1 gene expression (mean IL-1/RPL32 ratio) was 0.0919, 0.1357, 0.1527 and 0.2274 in the MPC:THP-1 cell ratio groups of 0:1, 1:50, 1:20 and 1:10, respectively. Percentage increase in IL-1 gene expression following LPS stimulation were 1294.5, 2116.7, 1198.6 and 1065.3% in the MPC:THP-1 cell ratio groups of 0:1, 1:50, 1:20 and 1:10, respectively. Conclusion: These results indicate that MPCs can modulate pro-inflammatory gene patterns in monocyte lineage

Poster Presentations

cells, with the degree of modulation being dependent on relative MPC quantity. Further studies will aim to evaluate the anti-inflammatory effects of MPC on cardiovascular disorders driven by exuberant monocyte activation. doi:10.1016/j.hlc.2007.11.133 Cost-effectiveness of screening BNP-levels in patients atrisk of asymptomatic left ventricular dysfunction Ella Zomer a,∗ , Danny Liew a , Bert Boffa b a NHMRC Centre for Clinical Research Excellence in Therapeutics, Department of Epidemiology & Preventive Medicine, Monash University, Australia b Health Benefits Australia, Australia

Purpose: We sought to evaluate the cost-effectiveness of screening ‘high-risk’ patients for (asymptomatic) left ventricular dysfunction (LVD) with brain natriuretic peptide (BNP), and subsequent treatment of those identified with LVD with enalapril.

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with asymptomatic LVD’, ‘Alive with (clinically manifest) heart failure’ and ‘Dead’. The annual risks of developing heart failure and death among people with asymptomatic LVD, and the annual risks of hospitalization and death among people with heart failure, were calculated from the Studies of Left Ventricular Dysfunction (SOLVD). Costs to the health sector comprised those associated with screening, drug treatment and hospitalization for heart failure, and were sourced from a prospective, longitudinal study conducted by Liao and colleagues (2006). Results: At a 5-year time horizon, the incremental costeffectiveness ratios (ICERs) of a BNP-based screening strategy in terms of dollars per life year gained were $40,306, $28,767 and $25,414 for populations with 10%, 20% and 30% underlying prevalences of asymptomatic LVD, respectively. ICERs decreased with increasing time horizons and an increasing underlying risk of LVD among the target population. Abstract table

Underlying prevalence of asymptomatic LVD Years to model

0.1

0.2

0.3

5

$40,306

$28,767

$25,414

($16,126–$212,825)

($12,289–$165,579)

($8,257–$180,132)

$28,484

$21,345

$19,577

($13,761–$134,460)

($9,346–$128,511)

($8,221 - $82,671)

$21,671

$16,754

$14,979

($11,080–$102,735)

($7,829–$68,922)

($6,735–$74,126)

$17,424

$13,591

$12,217

($9,341–$77,245)

($6,730–$54,686)

($5,783–$45,303)

$14,836

$11,395

$10,274

($7,523–$46,963)

($5,767–$45,272)

($4,980–$37,030)

$12,380

$9,844

$8,850

($6,920–$43,856)

($5,031–$31,115)

($4,485–$31,776)

6 7 8 9 10

Methods: A Markov-based decision analytic model was constructed with three health states in each arm: ‘Alive

Conclusions: Screening for asymptomatic LVD and subsequent treatment with enalapril represents a costeffective means of reducing heart failure-associated morbidity and mortality. doi:10.1016/j.hlc.2007.11.134

POSTER PRESENTATIONS

Heart, Lung and Circulation 2008;17S:S4–S53