|Home | About | Journals | Submit | Contact Us | Français|
Heparin (HP) inhibits pulmonary artery smooth-muscle cell (PASMC) growth in vitro and vascular remodeling in vivo. Barzu et al (1994) suggested that the antiproliferative effect of HP on rat aortic smooth muscle cell in vitro diminishes with prolonged exposure to heparin. We exposed cultured bovine PASMC (BPASMC) to prolonged pretreatment with 20μg/ml of 0- hexanoylated HP from passages 3 to13 and compared them to control (no pretreatment) cultures of identical passages. The pretreated BPASMC and control groups were growth arrested for 48 hrs, followed by treatment of 0- hexanoylated HP at different doses. On day five, the growth inhibition of BPASMC was determined. The percent inhibition by 1μg/ml of 0- hexanoylated HP was 46±14% vs. 62±13%, for control and pretreated BPASMC respectively. At 10μg/ml the inhibition was 62±7% vs. 84±6%. For 100μg/ml the inhibition increased to 92±5% vs. 100% and at 200μg/ml the inhibition was 95±3% vs. 100%. BPASMC (with or without preexposure to 0- hexanoylated HP), at passage 13, were sensitive to the growth inhibitory effect of 0- hexanoylated HP with no significant difference among the groups (95±3% inhibition vs. 100% for pretreated BPASMC). We found that 0-hexanoylated HP induced necrosis as shown by flow cytometry and only minor apoptosis. Caspase3 and PARP detection was insignificant between the groups. In summary, no cell subpopulation at long-term treatment exhibited resistance to 0- hexanoylated HP. The HP antiproliferative effect on SMC is potentially important in defining new approaches to the treatment of the remodeled vasculature of pulmonary hypertension.
The pathological feature of pulmonary hypertension is an increased medial thickening of the pulmonary artery due to hypertrophy and hyperplasia of pulmonary artery SMC (PASMC) (Jeffery et al., 2001; Rabinovich, 2004). Vascular smooth muscle cell (VSMC) proliferation has long been considered to be a key point in the remodeling of the vascular wall following vascular injury. Heparin inhibits (VSMC) growth both in vivo and in vitro (Clowes and Karnovsky, 1977; Hoover et al., 1980; Khoury and Langleben, 2000); the mechanisms responsible for the anti proliferative effects of heparin are not well known. Previous studies have shown that antiproliferative heparins significantly inhibit pulmonary vascular remodeling induced by hypoxia in rodents(Hales et al., 1983; Garg et al., 2000) and PASMC proliferation in culture (Thompson et al., 1994; Cindhuchao et al., 2003). Barzu et al. (1994) have suggested that long term treatment of SMC with heparin selected SMC with low sensitivity to the growth inhibition by heparin over multiple passages. Thus San Antonio et al. (1993) concluded that rat VSMC population during continuous passage may give rise to heparin resistant cells under selective conditions. Moreover, arterial SMC from explants of restenotic lesions have been shown to exhibit a decrease in growth inhibiton by heparin and to grow even more rapidly than control vessel (Chan et al., 1993). The results of Caleb et al. (1996) suggested that an altered responsiveness of SMC to heparin is dependent upon the expression of oncogenic proteins in the cells. In this study, we analyzed the antiproliferative effect of heparin on BPASMC during a long term treatment extending to passage 13 with different heparin concentrations and we looked for any resistance to HP during the long term of treatment. The heparin we used is hexanoylated which decreases the anticoagulation capacity of the heparin without decreasing its antiproliferative potential, allowing it to be used in larger doses than native heparin.
Bovine pulmonary arteries were ordered from a slaughterhouse. The method of Russell. (1971) had been applied for the isolation and culture of SMC. BPASMC were then harvested according to the protocol and kept in vials in liquid nitrogen. Frozen cells at passage two were thawed and grown to confluence in RPMI 1640 containing L-glutamine standard media (BioWhittaker, Walkersville, MD), with 10% FBS (BioWhittaker, Walkersville, MD) supplemented with 10,000 U/ml penicillin and 10,000 μg/ml streptomycin per ml (BioWhittaker, Walkersville, MD), and 250 μg/ml amphotericin B (Gibco, Grand Island, NY). The cells were incubated at 37 °C in air and 5% CO2. Cells from passage three to thirteen were used in these experiments.
HP fragments were Prepared by periodate treatment of HP, followed by sodium borohydride reduction. The tributylammonium salt of this fragmented HP has 0-acylated with hexanoic anhydride to give 0-hexanoylated low molecular weight HP as described by us earlier (Garg et al., 2006).
The isolated BPASMC were seeded at 1.25×104 cells/well into six-well tissue culture plates (Multiwell 353046; Becton Dickinson Labware, NJ) containing 2 ml of RPMI-1640 standard media with the antibiotic supplements and 10% FBS. The cell culture were run in sextuplicate for each determined dose of HP, three times. After 48 h, the cells were growth-arrested by decreasing the serum concentration of the cultured media to 0.1%. Following 48 h of the growth-arrested phase, all cells were fed 10% FBS and either treated with different concentrations of 0- hexanoylated HP (1, 10,100 and 200 μg/ml) or a control with no treatment. To obtain HP treated SMC (HT-BPASMC); cultures were permanently exposed to 20 μg/ml 0-hexanoylated HP, starting at the third passage and during thirteen subsequent passages. The HT-BPASMC were compared with control cultures of identical passage (run in parallel in the absence of preexposure to HP). On the fifth day, cells were harvested by trypsin-EDTA and counted under confocal microscope. Percent inhibition was calculated using the following relation:
Analysis of apoptosis was performed using the Annexin-V-FITC staining kit (BD Pharmigen, San Diego, CA). Cells were trypsinized, washed with PBS and suspended in 100μl staining solution (Annexin–V-FLUOS/Propidium Iodide). Following 15 min incubation at room temperature, 10000 cells were analyzed using the flow cytometry FACScan. Necrotic cells were differentiated from the Annexin-V positive cells by double staining with Propidium Iodide.
Proteins were extracted by suspending the cells in extraction buffer containing proteases and proteins inhibitors. Cells were then sonicated for 20 s on ice and centrifuged at 10 000 g for10 min at 4°C. Protein concentrations were quantified according to the Bradford protein assay protocol. A total of 20 or 25 μg of protein were loaded in each lane for western blot analysis. The proteins were then transferred to a nitrocellulose membrane by electro-blotting. After washing, the membrane was incubated with primary antibody cleaved caspase-3(rabbit IgG, 1:10000 in PBS-T and 5% BSA) for one hour, followed by incubation with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase at room temperature for 1 h (1:10000 in PBS-T, 5% BSA). After washing, the membrane was incubated with chemiluminescent substrate for 5 min and then exposed to a film. To control for variation in loading, the expression of housekeeping gene GAPDH was also assayed by western blot analysis.
Cleaved PARP was measured in the supernatant of cell’s homogenate from different groups of heparin treatment concentrations, using a commercially available ELISA kit containing antibodies that were cross-reactive with bovine cleaved PARP (Novus Biologicals, LLC, Littleton, CO, 80160 USA). Each sample was run in triplicate according to the protocol provided by the manufacturer.
The preparation of cell extracts, electrophoresis and transfer were carried out exactly as described earlier for cleaved caspase-3 using PCNA and cleaved PARP primary antibodies. Afterwards the blots were stripped and stained with GAPDH antibody (1:10000, Cell signaling, MA, USA). The results of Western blots were quantified with an image analyzer to measure the density of each band of interest.
All values were expressed as means ± SEM. Statistics were done using the computer program Stat view (SAS Institute Inc., Cary, NC) with factorial ANOVA. If ANOVA were significant, multiple comparisons were made using Fisher’s protected least significant difference test.
HT-BPASMC and control SMC in an equivalent passage were seeded and grown during 13 subsequent passages. The HT-BPASMC but not the control SMC, were cultured in the presence of 20μg/ml of hexanoylated heparin in the media. Then their sensitivity to the antiproliferative activity of 0-hexanoylated HP was assessed and compared to the sensitivity of control SMC of the same passage. The HT-BPASMC showed more sensitivity to HP inhibition over different passages. No resistance to HP was found over the 13 passages. (Figure 1)
The content of apoptotic and necrotic SMCs was compared in culture with different concentrations of 0-hexanoylated HP; using staining with Annexin-V-FLUOS and Propidium Iodide for flow cytometry analysis. The number of apoptotic cells in growing cell cultures with 10% FBS and 1 μg/ml HP was 12.07% where 21.28% were necrotic. In the presence of 10 μg/ml HP, 8.81% of SMCs were apoptotic and 27.80% were necrotic. In cultures with 100 and 200 μg/ml HP, 7.65% and 5.41% were apoptotic, 35.10% and 33.53% were necrotic respectively. (Figure 2)
Apoptosis was analyzed using a Western blot Cleaved Caspase-3 antibody. We choose to use this assay because caspase-3 is central to the caspase cascade and is activated by many apoptotic stimuli. The four SMC cultures were treated with 10% FBS in the presence of 1, 10,100 and 200 μg/ml of HP respectively. There was no difference between control cells of caspase-3 and cleaved caspase-3, and between different groups at different concentrations. (Figure 3)
BPASMC were treated with 1, 10,100 and 200 μg/ml of 0-hexanoylated heparin. An ELISA was run on the supernatant of homogenates which results showed no significant difference between the control and the groups treated with different doses of heparin. (Figure 4)
BPASMC were treated with 0-hexanoylated HP at different concentrations. No significant difference in protein expression of cleaved PARP was noticed between the control and heparin 1, 10,100 μg/ml treated cells. A decrease of cleaved PARP at heparin 200 μg/ml versus control was significantly different. (Figure 5)
After 5 days of 0-hexanoylated HP treatment at a concentration of 1, 10,100 or 200 μg/ml, the BPASMC showed a decrease in PCNA consistent with a decrease in proliferation by heparin in a dose dependent manner. (Figure 6)
The growth characteristics of BPASMC cultures suggested that long term heparin treatment showed no resistance over 13 passages (Figure 1). The number of apoptotic and necrotic SMC’s in culture showed necrosis and minor apoptosis (Figure 2). The level of cleaved caspase 3 did not show any difference among groups and control (Figure 3) and HP had no significant effect on cleaved PARP (Figure 4, Figure 5) but had a significant decrease in the PCNA expression in SMC (Figure 6). These results support that heparin effect on SMC’s is more likely through necrosis rather than apoptosis. In a normal artery, the VSMC are in a non-proliferative state and have a differentiated phenotype. After vascular injury, there is a loss of this phenotype and a switch to a synthetic phenotype leading to migration and proliferation (Zargham, 2008). Pulmonary vascular remodeling is a major contributor to the elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension. Smooth muscle cell (SMC) proliferation is a key event during the formation of intimal hyperplasia that contributes to vascular lumen narrowing (Aldons, 2000; Bennett and O’Sullivan, 2001). SMC function can be regulated by HP and has been shown to be effective in vitro and in vivo (Clowes and Karnovsky, 1977; Jurgen et al., 1998). Previous studies have shown that SMC from arteries exhibited a heterogeneous cell population. After isolating a subset of SMC, Schwartz et al, (1990) suggested that this subpopulation was most likely involved in the intimal hyperplasia and this fraction of cells was less sensitive to growth inhibition by HP (Clowes and Clowes, 1985). This decrease of sentivity to growth inhibition to HP has been confirmed with SMC obtained from explants of restenotic lesions (Chan et al., 1993). Therefore Pukac et al. (1991) and San Antonio et al. (1993) got heparin resistant rat aortic SMC by cloning and growing of cells over 30 passages with HP. Furthermore, Barzu et al. (1994) showed that long term HP treatment of rat aortic SMC over 13 passages resulted in significant decrease of sensitivity of SMC to the antiproliferative effect of HP. Results obtained in our study showed that BPASMC cultured over 13 passages and exposed chronically to 20μg/ml of HP exhibited no resistance over time to the growth inhibition by HP. The pretreated BPASMC at 20 μg/ml of HP showed even more sensitivity compared to the nonpretreated cells, Barzu et al. (1994) used a high dose of HP of 200μg/ml but when we tried this dose on our BPASMC they all died. We also grew rat aortic SMC which Barzu et al grew, but in 200μg/ml of HP they stopped growing at Passage 6. The different results may reflect the heterogeneous populations of SMC (Halayako and Stephens, 1994); the age of the animal from which the SMC are extracted and the duration of time of cells in culture. The purity of the HP used also may cause the cells to respond to the antiproliferative effect of HP with disparities. Moreover Ottlinger et al. (1993) emphasized that heparin-sensitive and insensitive pathway for arterial SMC proliferation may exist through inhibition by HP of MAPK pathway. The signaling pathways affected by the inhibitory effect of HP on SMC proliferation have recently been partially identified, it does involve the NA/H+ exchange and p27 gene as regulators of proliferation (Yu et al., 2005, 2006, 2008).
Besides growth inhibition, HP may induce death of SMC by apoptosis or necrosis. Previous studies have shown that HP does not induce apoptosis in pulmonary vascular pericytes and in porcine aortic SMCs (Khoury and Langleben, 2000; Pelisek et al., 2001). As assessed by complementary techniques, our data now provides evidence that 0-hexanoylated HP did not induce apoptosisin SMC. FACS analysis detected a significant increase in necrosis versus apoptosis and it was in dose dependent manner of HP concentrations. This was in accordance with Pelisek et al. (2001). Likely the inhibitory effect of HP is not mediated by a single cellular process and it seems likely that both necrosis and apoptosis are in effect with predominance of one effect to another. Most apoptosis signaling pathways ultimately result in caspase activation. After initiation caspase 3 is activated and PARP the caspase substrate is processed indicating that apoptosis pathway is activated (Friesen et al., 2008). To further elucidate the mechanism by which HP affected the inhibition of SMC, a quantitative analysis of cleaved caspase-3 activity indicated that HP did not show any difference between control cells and HP treated cells. This data is in support of Mason et al. (2003) stating that HP did not affect apoptosis in myometrial and leimyoma SMC. Also these results complement previous reports demonstrating that HP blocks cleavedcaspase-3 production induced by the activation of the TLR-2 in trophoblasts (Hills et al., 2006). Thus any maintenance of static Cleaved Caspase 3 by a heparin compound in vitro is unlikely to be through apoptosis (Khoury and Langleben, 2000). This biochemical data indicate that the non proliferation of cells in HT-BPASMC is unlikely because of induction of apoptosis. The apoptotic and necrotic cells death are accompanied by the cleavage of DNA and proteins. The cleavage of DNA repair associated nuclear enzyme poly (ADP-Ribose) polymerase PARP has become a hallmark for apoptosis in different types of cells responding to a wide range of apoptotic agents (Lazebnik et al., 1994). However, to our knowledge, no in vitro study has been reported to describe the correlation between heparin and SMC effect on cleaved PARP. In our study heparin is likely not having any effect on PARP. Taken together, these observations support the notion that heparin is acting most likely by inhibition of proliferation rather than through the apoptosis pathway. Proliferating cell nuclear antigen (PCNA) was identified as a component required for the in vitro replication machinery and repair. It’s also a well known marker of cell proliferation and a co-factor of DNA polymerase and has been shown to interact with cellular proteins involved in cell cycle regulation and check point control (Kelman, 1997). The polymerase delta accessory protein is present in cycling cells from late G1 to early G2 phase distinguished by its apparent association with cell division. The rate of cyclin synthesis is very low in quiescent cells and increases several fold after serum stimulation shortly before DNA synthesis. Subsequent work demonstrated that PCNA and cyclin are the same protein (Mathews et al., 1984). The fact that heparin decreased the PCNA expression in cultured SMC cells indicates that HP exerts inhibitory effects on the proliferation potential in these cells. Interestingly, the effect of HP on PCNA expression was dose dependent increasing with HP concentration. This was consistent with results obtained by Matsumoto et al. (2002) as evidenced by the reduced PCNA-positive on VSMC after HP treatment. Horiuchi et al. (1996), also showed suppression of PCNA after HP treatment in SMC supporting our findings.
The numerous phenotypes and different functions of vascular SMC subpopulations raise important issues as to why cellular dissimilarity prevails. Our results show that 0-hexanoylated heparin, a weakly anticoagulant but strongly antiproliferative heparin derivative, does not show the development of HP resistance with chronic exposure. 0-hexanoylated HP is thus a potential therapeutic agent for PASMC proliferation as occurs in hypertensive pulmonary arteries.
The authors thank John Beagle for its proofreading efforts. All authors declare that they have no competing interests. The present work was supported by NIH/NHLBI T32HL007874 and HL 39150 Grants.