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To investigate the involvement of apoptosis in the pathogenesis of T3-induced pulmonary hypertension in the broiler chickens, expression of caspases 1, 2 and 3 genes were evaluated by semi-quantitative RT-PCR in the lung and heart ventricles. Chicks were reared for 7 weeks under standard conditions. T3 as a Thyroid hormone was added to the ration after week one of rearing. Pulmonary hypertension was induced at 49 days based on RV/TV ratio index. The relative amount of caspases (1, 2 and 3) mRNA in the lung and right / left ventricles of heart was higher in T3-treated broilers than in control broilers at 14 and 49 days of age (P < 0.05). Within the control groups, caspases (1, 2 and 3) mRNA amounts did not significantly change in the lung and heart with increasing age. In contrast, in the treated groups, there was significant (P < 0.05) increase of caspases (1, 2 and 3) mRNA amounts according to age in the lung and ventricles of heart. It is speculation that increased mRNA levels of caspases in the lung and heart of pulmonary hypertensive broilers could be evidence of up-regulated apoptosis, which is involved in the pathophysiology of broiler chickens with pulmonary hypertension.
Key words: caspases, apoptosis, broiler, pulmonary hypertension, ascites
Pulmonary hypertension syndrome (ascites), is a metabolic disorder, characterized by hypoxemia, overload of the cardiopulmonary system, venous and heart congestion, right ventricle hypertrophy and a flaccid heart (1). Broilers are more sensitive to heart failure compared to other classes of chickens. Factors such as body performance, oxygen demands, hematological parameters and cellular interactions may all be involved in the resistance or susceptibility of broilers to heart failure and pulmonary hypertension syndrome (PHS) (2). This syndrome is the most conventional cardiomyopathy in industrial broilers (3). Many studies have been reported that different causes such as altitude, cold stress, lighting, air quality, ventilation, high nutrient density rations and incubator environment, have all been implicated in the development of pulmonary hypertension (4, 5).
The pathogenesis of pulmonary hypertension is a complicated, multifactorial process in which the combined effects of pulmonary artery vasoconstriction, vascular remodeling and thrombosis involve to a continuous high pulmonary vascular resistance (6-8). Vascular remodeling is associated with the low-grade in¬‚ammation, vascular ¬brosis and apoptosis(9).
Apoptosis, or programmed cell death, is a multi-pathway biological process, which contributes in many physiological and pathological phenomena. Multicellular organisms regularly eliminate and renew cells to maintain their homeostasis. Abnormal function of this process has been implicated in atherosclerosis, cancer, heart failure, and pulmonary hypertension (9, 10).
At the cellular and molecular levels, apoptosis is identified by morphological and biochemical changes such as cell shrinkage, formation of apoptotic bodies, caspase activation, chromatin condensation, DNA fragmentation (9, 11). Many molecular processes of apoptosis are mainly mediated by specific cysteine proteases called caspases. Caspases are enzymes with a crucial cysteine residue that can have proteolytic activity and cleave other subcellular cytoplasmic proteins at aspartic acid residues and fragment nuclear DNA. The name of caspases derives from its specialized function: cysteine-aspartic acid-proteases (12). Ten major caspases have been determined as following: initiators (caspases 2, 8, 9 and 10), effectors or executioners (caspases 3, 6 and 7) and inflammatory caspases (caspases 1, 4 and 5). Other caspases have been reported such as caspase-11, a regulator of apoptosis and cytokine maturation in septic shock, caspase -12, a mediator of endoplasmic reticulum-specific apoptosis and cytotoxicity due to amyloid-ß , caspase-13, a cysteine protease identified in cattle, and caspase-14, a highly expressed caspase in embryonic tissues (13).
The objective of this study was to determine levels of caspases 1, 2 and 3 mRNA expression as candidates of inflammatory, initiator and effector caspases in the lung and right/left ventricles of heart in the pulmonary hypertensive broiler chickens induced experimentally by 3,5,3´-l-triiodothyronine (T3).
2. Materials and methods
Forty-two, one-day-old fast-growing chickens (Ross 308) were randomly divided into two equal groups with three replicates per group. Chicks were reared in the floor pens (wood shaving litter) for 7 weeks under standard conditions. A standard ration (Starter: 13 MJ ME/kg of diet, 230 g/kg crude protein (CP), grower: 13 MJ ME/kg of diet, 200 g/kg CP, finisher: 13 MJ ME/kg of diet, 180 g/kg CP formulated to meet requirements for broilers. Water was offered as ad libitum access. In the treatment group, T3 (Sigma Chemical Co.) was added to the ration (1.5 mg T3/kg) after week one of rearing (14). Mortality was daily recorded and dead broilers were inspected for lesions of heart failure and ascites.
2.2. Assessment of right ventricle Hypertrophy
At 14 and 49 days of age, 6 chicks from each group were selected at random, weighed and killed by decapitation. The heart was resected, and right ventricle / total ventricle ratio (RV/TV) was calculated as described by (14). If this ratio was more than 0.290, PHS has been occurred in chickens according to (15). The right and left ventricles plus lung were immediately frozen in the liquid nitrogen and stored at -70°C for subsequent RNA analysis.
2.3. RNA extraction of lung and heart tissues
Total RNA was extracted from lung and right / left ventricular tissues using TRIzol reagent (Invitrogen, Karlsruhe, Germany). Homogenized lung tissue (100 mg) was prepared in digestion buffer. The homogenate was mixed with chloroform. After centrifuging the mixture, total RNA settled in the upper aqueous phase. Following precipitation with isopropanol, the RNA pellet was rinsed with ethanol. The samples of RNA were resuspended in DEPC-treated water. To remove eventually residual DNA, the RNA was treated by DNase; then, the RNA was measured and qualified by spectrophotometry. Only RNA of sufficient purity, having an absorbance ratio (A260/280) greater than 1.9, was considered for synthesis of cDNA. It was analyzed by electrophoresis on a 2% agarose gel, stained with 0.5 m g/ml ethidium bromide.
2.4. Semi-quantitative reverse-transcription PCR
Expression of caspases (1, 2 and 3) along with the β-actin gene as housekeeping control was done by reverse-transcribed polymerase chain reaction (RT-PCR) using the SuperScript One-Step RT-PCR kit with Platinum Taq (Invitrogen, Karlsruhe, Germany). The primer sequences are listed in Table 1. Caspases primers were designed from their nucleotide sequences in Genebank (the accession numbers are offered in Table 1). For β-actin (used as a housekeeping gene) previously described primer pairs (14) were used. Reverse transcription was performed at 50°C for 30 minutes. The amplification step consisted of 24-27 cycles (leading to linear range of amplification): denaturation at 94°C for 40 seconds, annealing for 50 seconds at 60-64°C, and extension of primers for 50 seconds at 72°C. The products were then held at 72°C for 5 minutes for DNA extensions to occur. Amplified fragments of caspases and β-actin cDNA were separated by electrophoresis on 1.5% agarose gel, and the products were visualized by staining with 0.5 μg/mL ethidium bromide. Density of bands was determined using Photo-Capt V.99 Image software, and relative densities were expressed as caspases (1, 2 and 3) / β-actin density.
2.5. Statistical analysis
All results are represented as mean ± SEM. The statistical analysis was carried out using SPSS 14.0 software (SPSS Inc., New York, USA). Comparisons were made between control and T3-treated groups at the same age using student-T test. Differences were considered significant at P < 0.05.
3.1. Assessment of right ventricular hypertrophy
RV/TV ratio was measured as an index of right ventricular hypertrophy and pulmonary hypertension (15).The amounts of this index in treated and control groups in 14 days were 0.161 ± 0.018 and 0.181 ± 0.011 respectively; while in 49 days were 0.242 ± 0.013 and 0.30 ± 0.010. As noticed, the increasing of RV/TV ratio was only significant (P < 0.05) in the treated group in 49 days of age (25%).
3.2. Effect of T3 on Caspases (1, 2 and 3) mRNA expression in the lung and heart
Expression of caspases (1, 2 and 3) gene was studied using semi-quantitative RT-PCR in the lung and right/ left heart ventricles of pulmonary hypertensive broilers (induced by T3) at two ages (14 and 49 days). Reverse transcription-PCR results are shown in Fig. 1-4. The expression of β-actin was detected which was consistent for all groups. The caspases (1, 2 and 3) genes were expressed in the lung and the right/left ventricles of control and T3-treated broilers at 14 and 49 days of age. The relative amount of caspases (1, 2 and 3) mRNA expression in the lung and right / left ventricles of heart was significantly (P < 0.05) higher in T3-treated broilers than in control broilers at 14 and 49 days of age. The increasing amounts of caspase-1 were 54% and 125% in the lung, 32% and 88% in the left ventricle and 39% and 147% in the right ventricle at 14 and 49 days of age, respectively (Fig. 1). The increasing amounts of caspase-2 were 84% and 130% in the lung, 50% and 183% in the left ventricle and 53% and 104% in the right ventricle at 14 and 49 days of age, respectively (Fig. 2). The increasing amounts of caspase-3 were 86% and 143% in the lung, 34% and 106% in the left ventricle and 89% and 149% in the right ventricle at 14 and 49 days of age, respectively (Fig. 3).
In the control groups, caspases (1, 2 and 3) mRNA amounts did not significantly change in the lung and heart with increasing age. In contrast, in the treated groups, there was significant (P < 0.05) increase of caspases (1, 2 and 3) mRNA amounts according to age in the lung and ventricles of heart (Fig. 1-3). The increasing amounts of caspase-1 were 24% in the lung, 62% in the left ventricle and 64% in the right ventricle between 14 and 49 days of age, within treated groups (Fig. 1). The increasing amounts of caspase-2 were 19% in the lung, 129% in the left ventricle and 61% in the right ventricle between 14 and 49 days of age, within treated groups (Fig. 2). The increasing amounts of caspase-3 were 13% in the lung, 83% in the left ventricle and 26% in the right ventricle between 14 and 49 days of age, within treated groups (Fig. 3).
This research evaluated mRNA levels of three types of caspases (i.e. caspases 1, 2 and 3) in the lung and left/right heart ventricles of pulmonary hypertensive broilers. According to Wideman (2001) (15) report, right ventricular hypertrophy is positively correlated with high pulmonary arterial pressure, and RV/TV ratio could be an evidence for PHS. Accordingly, in 49 days of rearing, this index (RV/TV ratio) confirmed T3-induced PHS. Thus, cell responses at 14 days of age were not because of PHS. In this age, T3 hormone probably involved in the cell responses and genes expression.
Molecular and cellular mechanisms of thyroid hormones have been determined but it is not well known in chickens. Upadhyay et al. (16) reported DNA fragmentation of liver cells in the hyperthyroid rats and suggested increasing of apoptosis. They showed that caspase-3 is increased in the hyperthyroid rats. Similar study was done by Wang et al. (17) who demonstrated an elevation in the apoptosis and DNA fragmentation of cardiomyocyte exposing to thyroid hormone. However, in our study there was predictable up-regulation of apoptosis in the T3-treated chickens (at 14 days of age) as determined by higher gene expression of caspases.
It is clear that apoptosis occurs under both physiological and pathological conditions. Then, detection of caspases mRNA in the heart and lung of intact and pulmonary hypertensive chickens had been expected in the all ages. There are numerous modulators of apoptosis in the vasculature system that makes apoptosis as a complex cascade. Reactive oxygen species NO, angiotensin type 2 (AT2), receptors and the endothelin system are examples of these modulators. Reactive oxygen species are an important factor in the pathogenesis of pulmonary hypertension and in apoptosis. It has been shown that O2 could induce proliferation and H2O2 enhance apoptosis via a protein kinase C-dependent mechanism (9). It has been also determined that a progressive hypoxia developing in chickens with pulmonary hypertension can cause increased reactive oxygen species (ROS), and production of ROS influences the metabolic systems including cardiac systems in pulmonary hypertensive birds (1, 18, 19). Another apoptotic modulator is nitric oxide. Several studies reported that contrary to mammals, nitric oxide in chickens is reduced during pulmonary hypertension (20, 21). However, this factor may also influence apoptotic process (22). Hassanpour et al. (23) suggested that higher endothelin of serum in the broiler chickens versus layer chickens make broiler chickens to be more susceptible to endothelin related-cardiomyopathies such as heart failure and pulmonary hypertension. They also confirmed that serum endothelin is increased in the pulmonary hypertensive chickens compared to healthy chickens (14). It is possible that this variation in the endothelin system of broiler chickens affects apoptosis.
The present study revealed higher expression of apoptosis-related genes (i.e. Caspases) in the heart and lung of pulmonary hypertensive chickens, which could support high apoptosis in those tissues. Apoptosis as a mechanism for the progression and development of heart failure has been implicated by several studies (24-26). apoptosis is also considered to cause vascular remodeling in the pulmonary hypertension (9, 27). However, these studies confirmed the presence of high apoptosis in accordance to our results.
In conclusion, our data show that caspases 1, 2 and 3 genes are normally expressed in the lung and heart ventricles of broilers. It is probably that increased mRNA levels of caspases in the lung and heart of pulmonary hypertensive broilers could be evidence of up-regulated apoptosis, which is involved in the pathophysiology of broiler chickens with pulmonary hypertension. However, this study is the first step for future studies; and other methods such as immunohistochemistry would be helpful to confirm enhanced apoptosis in the PHS.
This work was supported by funds granted by the Applied Research Centre, Vice Chancellor for Research of Islamic Azad University, Science and Research Branch.