Effect Of PKD1Downregulation On The Pathogenesis Of Aortic Dissection

Aortic dissection (AD) is a catastrophic event involving the aorta with high mortality. In recent years, the prevalence of AD has been increasing in the world, which leads to high consumption of medical resources and social wealth. Although some factors are linked with AD formation such as hypertension, atherosclerosis and smoking et al, few persuasive etiological hypotheses has been proposed. Thus the pathogenesis of AD remains elusive.Extensive pathological studies have observed that smooth muscle cell (SMC) rarefaction, extracellular matrix (ECM) disorders and matrix metalloproteinase (MMP) overexpression are the common histological features of AD. Hence it is hypothesized that compromised structural integrity of aortic wall may be the triggering factor of AD formation. Based on the weakened aortic wall, Hypertension and other related risk factors predispose to initiating dissection.Polycystic kidney disease1gene (PKD1) is the pathogenic gene of autosomal dominant polycystic kidney disease. PKD1encodes polycystin-1, which is a large cell-surface protein involved in cell-cell/matrix interactions and cell signaling. As revealed by PKDl knockout mice, PKDl downregulation resulted in a series of pathologic alterations in aortic walls of those mice such as latent lacouna and intramural hematoma, which are similar with human AD. Our preliminary research also observed that expression of PKDl was significantly downregulated in the aortic media of AD patients. In this study, we further explore the interaction among PKD1downregulation, SMC phenotypic switching and ECM disorders both at tissue and cellular level. This interaction can illuminate the effect of PKD1downregulation on the pathogenesis of AD and contribute to prevention, diagnosis and treatment for AD.Part I In vivo study of SMC phenotypes and ECM components in dissected aortic mediaObjectiveTo observe and evaluate diversities of SMC phenotypes, ECM components and MMP expression between dissected and affected aortic media.Methods18dissected thoracic aortic samples were collected from patients with AD and18unaffected thoracic aortic samples were obtained from age-and gender-matched controls. Expression of SMC phenotypic markers in the aortic media was evaluated by immunohistochemical staining and immunoblotting. The medial SMC ultrastructure was observed with transmission electron microscope (TEM). Special staining and immunoblotting evaluated ECM production and MMP expression in the dissected and affected aortic media.ResultsCompared with the unaffected aortic media:(1) Immunohistochemical staining and immunoblotting showed that, proteins levels of smooth muscle a-actin (a-SMA), smooth muscle-myosin heavy chain2(SM-MHC-2), Smoothelin and Calponin were significantly decreased (P<0.01for all) while those of osteopontin (OPN) and non-muscle myosin heavy chain B (SMemb) were significantly increased (P<0.01for both) in the dissected aortic media.(2) Results of TEM demonstrated that, in the dissected aortic media SMCs changed their typical elongated, spindle-shape, with contractile structures disappeared and synthetic organelles increased in the cytoplasm.(3) Masson’s trichrome and Gomori’s staining showed collagen fibers extensively deposited in the dissected media, while elastic fibers strikingly disrupted or decreased. Immunohistochemical staining further revealed the elevated deposition of collagens I and Ⅲ, which was consistent with immunoblotting results.(4) Immunoreactivities for MMP-2were increased in the dissected media (P<0.01). The staining for MMP-1was weak both in the unaffected media and in the dissected media (P>0.05). In vivo protein levels of MMP-2were higher in the dissected media (P<0.01), whereas there was no significant difference in that of MMP-1between the dissected and affected aortic media (P>0.05).ConclusionsIn the dissected aortic media, SMC have manifested phenotypic switching from the contractile to the synthetic type. Together with SMC phenotypic alteration, elastin degradation, collagen deposition and MMP-2overexpression are also observed in the dissected media.Part Ⅱ In vitro isolation, primary culture and preliminary identification of thoracic aortic medial SMCsObjectiveTo isolate and culture SMCs derived from the dissected and affected aortic media with simple and effective method.Methods 18dissected thoracic aortic samples and18unaffected thoracic aortic samples were evenly divided into three groups,6pathologic samples and6control samples in each group. Then the three group samples were used to obtain medial SMC primary cultures with enzymatic digestion method, explant method and optimized explant technique respectively. The purity of SMC cultures was identified with immunocytochemical staining.Results(1) As for control SMC primary cultures from the unaffected media, the cultivation efficiencies of enzymatic digestion method, explant method and optimized explant technique were50%(3/6),100%(6/6) and83.3%(5/6) respectively. As for SMC primary cultures from the dissected media, the efficiencies of explant method and optimized explant technique were66.7%(4/6) and83.3%(5/6) respectively. It failed to obtain the dissection cultures using enzymatic digestion method.(2) For both control and dissection primary cultures, the cultivation period of enzymatic digestion method, explant method and optimized explant technique were5-6days,10-12days and4weeks respectively.(3) Whatever the culture method, control SMCs uniformly displayed an elongated, spindle-or triangle-like shape and appeared as the "hills and valleys" arrangement when cells reached confluence. Although the majority of SMCs from the dissected media showed a parallel appearance as control cells, some oval and rhomboid cells with porous cytoplasm were observed in the primary cultures. After passaged, there were also atypical-shape cells in the dissection subcultures.(4) In all three groups, the immunopositive proportions of control SMC cultures for a-SMA are more than95%, while less than10%for OPN. On the contrary, in explant group and optimized explant group, the immunopositive proportions of dissection cultures for a-SMA are16±2.6%and15±3.1%(P>0.05) respectively, while85±4.3%and88±1.7%(P>0.05) for OPN respectively. Both control and dissection SMC cultures had no staining for vWF or CD90/Thy-1.ConclusionsCompared to the traditional enzymatic digestion and explant method, optimized explant technique is a simple, convenient and efficient method to acquire SMC primary cultures from both the dissected and unaffected aortic media. The SMC primary cultures can be passaged stably in vitro without contamination of endothelial cells and fibroblasts. The dissection cultures show the atypical cellular morphology in contrast with control cultures. Part III In vitro study of association of SMC phenotypic switching with ECM disorders in aortic dissectionObjectiveTo identify and evaluate diversities of SMC phenotypes, ECM synthesis and MMP production between dissection SMC cultures and control cultures.MethodsUsing optimized explant technique,6dissection SMC cultures and6control cultures were obtained from6dissected aortic media and6unaffected media. Cell Counting Kit-8(CCK-8) assay and [3H] thymidine incorporation were used to evaluate in vitro proliferation of SMCs. The mRNA and protein levels of the phenotypic markers in the cultured SMCs were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescent staining and immunoblotting. In vitro ultrastructure of SMC was also observed with TEM.[3H] proline incorporation assay was used to assess collagen synthesis of the cultured SMCs. Immunoblotting was used to evaluate MMP production of SMC cultures.ResultsCompared with control SMCs:(1) The dissection SMC cultures exhibited significantly increased growth rates and DNA synthesis (PO.Olfor both).(2) In vitro mRNA and protein levels of a-SMA, SM-MHC-2and Smoothelin were also significantly reduced in the dissection cultures (P<0.0lfor all), while those of OPN were markedly increased (P<0.01).(3) In the cytoplasm of dissection SMCs, contractile structures including myofilament, dense body and dense patch disappeared, while the synthetic organelles such as endoplasmic reticulum and Golgi body increased and dilated.(4) The amounts of the newly synthesized collagens I and III were remarkably increased in the dissection cultures (P<0.01).(5) In vitro protein levels of MMP-2were higher in the dissection cultures (P<0.01), whereas there was no significant difference in that of MMP-1between the two group cultures (P>0.05).ConclusionsIn line with the findings of in vivo study, in vitro cultured SMCs from the dissected aortic media also exhibit the phenotypic switching from the contractile to the synthetic type. Besides, SMC phenotypic switching promotes collagen synthesis and MMP-2production.Part IV In vivo and in vitro study of the effect of PKD1downregulation on SMC phenotypic switching ObjectiveTo in vivo and in vitro examine PKD1expression in the dissected and unaffected medial SMCs, and evaluate the effect of PKD1downregulation on SMC phenotypic switching, collagen synthesis and MMP-2production.MethodsIn the media of18pathologic samples and18control samples, PKD1expression was evaluated by immunohistochemical staining and immunoblotting, and4dissection SMC cultures and4control cultures were obtained from those samples using optimized explant technique. The mRNA level of PKD1and the protein level of polycystin1in the cultured SMCs were analyzed by real-time RT-PCR and immunoblotting. Besides, the endogenous PKD1of control SMC was depleted by RNA interference. In addition to detect the depletion efficiency, pre-and post-interference diversities in phenotypic protein expression, collagen synthesis and MMP-2production of control SMCs were also analyzed.Results(1) Compared with controls, PKD1expression in the dissected medial SMCs were significantly decreased in vivo and in vitro (P<0.01for both).(2) After RNA interference, in vitro mRNA and protein levels of α-SMA, SM-MHC-2and Smoothelin were significantly reduced (P<0.01for all), while those of OPN were markedly elevated (P<0.01for both).(3) The amounts of the newly synthesized collagens Ⅰand Ⅲ were remarkably increased in PKD1-depleted SMCs (P<0.01for both).(4) In vitro protein levels of MMP-2were elevated in PKD1-depleted SMCs (P<0.01).ConclusionsCompared with the unaffected medial SMCs, PKD1expression is significantly downregulated in the dissected medial SMCs. After depleting the endogenous PKD1of the unaffected medial SMCs in vitro, they also exhibit the phenotypic switching from the contractile to the synthetic that is similar with the dissected medial SMCs. Therefore, through initiating SMC phenotypic switching, PKD1downregulation can promote ECM disorders and MMP overexpression, which impair the structure and the mechanical properties of the aortic wall and contribute to AD formation.