The Early Effects Of Shear Stress On Pulmonary Microvascular Endothelial Cells

BACKGROUNDCongenital heart disease (CHD) is one of the most common children’s congenital malformations, the incidence of which is about6%o-10%o. About1.5million newborn are born with CHD every year,60%～70%of which are left-to-right shut type. The left-to-right shut CHD can cause the blood flow of the pulmonary circulation increase and lead to pulmonary arterial hypertension (PAH), which is the common complication of CHD.4%-15%CHD patients will progress to PAH, the severity of which influences the development and prognosis of CHD directly.PAH is still a progressive disease characterized by neointimal formation, obliteration, and plexiform lesions. The underlying mechanism of which is still poorly understood. Endothelial cells (ECs) are flat cells lining on the intimal surface of the blood vessel, located between the blood flow and smooth muscle cells (SMCs), which play an important role in maintaining homeostasis. When the left-to-right shut occurs in CHD, the pulmonary blood flow will be increased and the mechanical stress, especially shear stress, exerted on the ECs will be changed, which will lead to the dysfunction of the ECs and result in the pulmonary vascular remodeling and PAH finally. The increasing shear stress is regarded as the initial factor of the PAH secondary to the left-to-right shut CHD, and ECs act as the direct sensor of shear stress play an vital role in the pathogenesis of PAH. For the heterogeneity of ECs, the distal pulmonary arteriole endothelial cells (PAECs) are more vulnerable to damage, the dysfunction of which is much more important in the early disease. Therefore, we select pulmonary microvascular endothelial cells (PMVECs) as the research object to reveal the truly essence of the PAH secondary to CHD.The research focus on the pathogenesis of PAH has shifted from the static research to the dynamic research mimic human physiological environment, from vasoconstriction mechanism to a cancer-like mechanism of angioproliferation. Researches have proved that endothelial cells apoptosis and dysfunction is the initial effect of the high blood flow pulmonary hypertension and put forward the "apoptosis trigger" hypothesis. Therefore, our researches focus on the early effects of shear stress on the cytoskeleton of PMVECs and the early effects of shear stress on the apoptosis of the PMVECs to explore the early pathogenesis of high blood flow PAH.Objective1. To observe the effects of shear stress on the cytoskeleton of PMVECs.2. To observe the early effects of shear stress on the apoptosis of PMVECs and the expression of anti-apoptotic gene BCL-2.Methods1. Rat PMVECs were cultured in ECM medium with conventional methods and were identified with CD31immunofluorescent staining.2. Self-made multi-layer chamber, together with constant flow pump, liquid accumulator and silicone tube to assemble the perfusion system.3. Exerted different shear stresses(0dyn/cm2、1dyn/cm2、1.75dyn/cm2、2.5dyn/cm2) on the PMVECs with different exerting time(0h、0.5h、1h、2h、4h), and observed the changes of the cytoskeleton with the F-actin immunofluorescent staining.4. Observed the apoptosis of different groups with the Annexin V/PI staining.5. Real-time PCR and Western blot were performed to measure the relative expression of BCL-2in the mRNA level and protein level respectively.6. Datum were analyzed by the SPSS17.0, and the datum were expressed in x±s, ANOVA test was used for comparison between groups. Value of P<0.05is considered as significant difference.Results1. The Immunofluorescent staining of F-actin in PMVECs after the treatment of different shear stresses at different time pointsMicrofilament rearrangement occurred in PMVECs due to the shear stress, changing from former disorderliness into orderliness towards the direction of the shear force. Stress fibers appeared along the cell long axis. This change was in a time and stress strength-dependent manner. With the extension of treatment time, the phenomena became more obvious, until nearly all the F-actin changed to be stress fibers.2. The apoptosis level of the different groups Flow cytometry analysis showed that the shear stress could induced the apoptosis of the PMVECs. The control group apoptosis level was lower to the experimental groups, and there were significantly differential between the groups. The apoptosis level would increase with the extending of the acting time.3. The expression of BCL-2gene in different groups The real-time PCR showed that the relative expression of BCL-2in the control group was higher than the0.5h group, but the expression would increase as the extending of the acting time, and there were significant difference between the groups. The western blot analysis showed that the relative protein expression of Bcl-2were consistent with the mRNA expression.Conclusions1. The cytoskeleton of the PMVECs will rearrange to be stress fibers according to the shear stress it endured. This phenomenon is both time and stress force-dependent, and occurs at the early stage. This may be closely related to the early pathogenesis of pulmonary hypertension induced by high pulmonary flow secondary to CHD.2. The shear stress can induce the apoptosis of PMVECs, and the effect will become more apparent as the extending of acting time of shear stress. The shear stress can lead to the decreased expression of the anti-apoptotic gene BCL-2at the beginning of the shear stress action. With the extending of the acting time, the BCL-2expression will be increased. The findings elucidate that the apoptosis of the endothelial cells may be the initial factors of the pathogenesis of the PAH, and with the extending of the shear stress acting time, the anti-apoptotic ECs were selected, which is consistent with the "apoptosis trigger" hypothesis of PAH.Innovations and significanceObserve the morphological and functional changes of PMVECs eliminating the influence of collagen and SMCs. Select the PMVECs as the research objective, and observe the early effect, mimic the early pathogenesis of the high blood flow PAH secondary to CHD. Develop the multi-layer flow chamber, which can batch the samples together, to ensure the same experimental conditions and make the experimental result more reliable.