Mesenchymal Cells Retain the Specificity of Embryonal Origin During Osteogenic Differentiation.

Mesenchymal stem cells (MSCs) are widely used in therapy, but the differences between MSCs of various origins and their ability to undergo osteogenic differentiation and produce extracellular matrix are not fully understood. To address this, we conducted a comparative analysis of mesenchymal cell primary cultures from 6 human sources, including osteoblast-like cells from the adult femur, adipose-derived stem cells, Wharton's jelly-derived mesenchymal cells, gingival fibroblasts, dental pulp stem cells, and periodontal ligament stem cells. We analyzed these cells' secretome, proteome, and transcriptome under standard and osteogenic cultivation conditions. Despite the overall similarity in osteogenic differentiation, the cells maintain their embryonic specificity after isolation and differentiation in vitro. Furthermore, we propose classifying mesenchymal cells into 3 groups: dental stem cells of neural crest origin, mesenchymal stem cells, and fetal stem cells. Specifically, fetal stem cells have the most promising secretome for various applications, while mesenchymal stem cells have a specialized secretome optimal for extracellular matrix production. Nevertheless, mesenchymal cells from all sources secreted core bone extracellular matrix-associated proteins. In conclusion, our study illuminates the distinctive characteristics of mesenchymal stem cells from various sources, providing insights into their potential applications in regenerative medicine and enhancing our understanding of the inherent diversity of mesenchymal cells in vivo.

Low dose cadmium inhibits syndecan-4 expression in glycocalyx of glomerular endothelial cells.

Cadmium (Cd) is one of the most polluting heavy metal in the environment. Cd exposure has been elucidated to cause dysfunction of the glomerular filtration barrier (GFB). However, the underlying mechanism remains unclear. C57BL/6J male mice were administered with 2.28 mg/kg cadmium chloride (CdCl2) dissolved in distilled water by oral gavage for 14 days. The expression of SDC4 in the kidney tissues was detected. Human renal glomerular endothelial cells (HRGECs) were exposed to varying concentrations of CdCl2 for 24 h. The mRNA levels of SDC4, along with matrix metalloproteinase (MMP)-2 and 9, were analyzed by quantitative PCR. Additionally, the protein expression levels of SDC4, MMP-2/9, and both total and phosphorylated forms of Smad2/3 (P-Smad2/3) were detected by western blot. The extravasation rate of fluorescein isothiocyanate-dextran through the Transwell was used to evaluate the permeability of HRGECs. SB431542 was used as an inhibitor of transforming growth factor (TGF)-ß signaling pathway to further investigate the role of TGF-ß. Cd reduced SDC4 expression in both mouse kidney tissues and HRGECs. In addition, Cd exposure increased permeability and upregulated P-Smad2/3 levels in HRGECs. SB431542 treatment inhibited the phosphorylation of Smad2/3, Cd-induced SDC4 downregulation, and hyperpermeability. MMP-2/9 levels increased by Cd exposure was also blocked by SB431542, demonstrating the involvement of TGF-ß/Smad pathway in low-dose Cd-induced SDC4 reduction in HRGECs. Given that SDC4 is an essential component of glycocalyx, protection or repair of endothelial glycocalyx is a potential strategy for preventing or treating kidney diseases associated with environmental Cd exposure.

Expression Pattern of Trace Amine-Associated Receptors during Differentiation of Human Pluripotent Stem Cells to Dopaminergic Neurons.

Trace amine-associated receptors (TAARs), which were discovered only in 2001, are known to be involved in the regulation of a spectrum of neuronal processes and may play a role in the pathogenesis of a number of neuropsychiatric diseases, such as schizophrenia and others. We have previously shown that TAARs also have interconnections with the regulation of neurogenesis and, in particular, with the neurogenesis of dopamine neurons, but the exact mechanisms of this are still unknown. In our work we analyzed the expression of TAARs (TAAR1, TAAR2, TAAR5, TAAR6, TAAR8 and TAAR9) in cells from the human substantia nigra and ventral tegmental areas and in human pluripotent stem cells at consecutive stages of their differentiation to dopaminergic neurons, using RNA sequencing data from open databases, and TaqMan PCR data from the differentiation of human induced pluripotent stem cells in vitro. Detectable levels of TAARs expression were found in cells at the pluripotent stages, and the dynamic of their expression had a trend of increasing with the differentiation and maturation of dopamine neurons. The expression of several TAAR types (particularly TAAR5) was also found in human dopaminergic neuron-enriched zones in the midbrain. This is the first evidence of TAARs expression during neuronal differentiation, which can help to approach an understanding of the role of TAARs in neurogenesis.

Modulation of Notch Signaling at Early Stages of Differentiation of Human Induced Pluripotent Stem Cells to Dopaminergic Neurons.

Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson's disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. Investigating the role of signaling cascades, important for neurogenesis, can help to solve this problem and to provide a deeper understanding of their role in neuronal development. Notch signaling plays an essential role in development and maintenance of the central nervous system after birth. In our study, we analyzed the effect of Notch activation and inhibition at the early stages of differentiation of human induced pluripotent stem cells to dopaminergic neurons. We found that, during the first seven days of differentiation, the cells were not sensitive to the Notch inhibition. On the contrary, activation of Notch signaling during the same time period led to significant changes and was associated with an increase in expression of genes, specific for caudal parts of the brain, a decrease of expression of genes, specific for forebrain, as well as a decrease of expression of genes, important for the formation of axons and dendrites and microtubule stabilizing proteins.

The Role of the Notch Signaling Pathway in Recovery of Cardiac Function after Myocardial Infarction.

Myocardial infarction (MI) is a pathological process, evidencing as massive death of cardiomyocytes associated with hypoxic and oxidative stress. The formation of areas of fibrosis ultimately leads to heart failure. There are some mechanisms that contribute to the functional repair of the heart. In most mammals, including humans, the Notch signaling pathway has cardioprotective effects. It is involved in the formation of the heart in embryogenesis and in the restoration of cardiac function after MI due to: (1) reducing oxidative stress; (2) prevention of apoptosis; (3) regulation of inflammation; (4) containment of fibrosis and hypertrophy of cardiomyocytes; (5) tissue revascularization; and (6) regulation of proliferation and differentiation of cardiomyocytes. In addition, the Notch signaling pathway interacts with other signaling cascades involved in the pathogenesis of MI and subsequent cardiac repair. In this review, we consider the Notch signaling pathway as a potential target for therapeutic approaches aimed at improving cardiac recovery after MI.

Dose-dependent mechanism of Notch action in promoting osteogenic differentiation of mesenchymal stem cells.

Osteogenic differentiation is a tightly regulated process realized by progenitor cell osteoblasts. Notch signaling pathway plays a critical role in skeletal development and bone remodeling. Controversial data exist regarding the role of Notch activation in promoting or preventing osteogenic differentiation. This study aims to investigate the effect of several Notch components and their dosage on osteogenic differentiation of mesenchymal stem cells of adipose tissue. Osteogenic differentiation was induced in the presence of either of Notch components (NICD, Jag1, Dll1, Dll4) dosed by lentiviral transduction. We show that osteogenic differentiation was increased by NICD and Jag1 transduction in a dose-dependent manner; however, a high dosage of both NICD and Jag1 decreased the efficiency of osteogenic differentiation. NICD dose-dependently increased activity of the CSL luciferase reporter but a high dosage of NICD caused a decrease in the activity of the reporter. A high dosage of both Notch components NICD and Jag1 induced apoptosis. In co-culture experiments where only half of the cells were transduced with either NICD or Jag1, only NICD increased osteogenic differentiation according to the dosage, while Jag1-transduced cells differentiated almost equally independently on dosage. In conclusion, activation of Notch promotes osteogenic differentiation in a tissue-specific dose-dependent manner; both NICD and Jag1 are able to increase osteogenic potential but at moderate doses only and a high dosage of Notch activation is detrimental to osteogenic differentiation. This result might be especially important when considering possibilities of using Notch activation to promote osteogenesis in clinical applications to bone repair.

Dysregulation of Notch signaling in cardiac mesenchymal cells of patients with tetralogy of Fallot.

BACKGROUND: Tetralogy of Fallot (TF) is a severe congenital defect of heart development. Fine-tuned sequential activation of Notch signaling genes is responsible for proper heart chamber development. Mutations in Notch genes have been associated with TF. The aim of this study was to analyze the activity of the Notch pathway in cardiac mesenchymal cells derived from ventricular tissue of TF patients. METHODS: Cardiac mesenchymal cells were isolated from 42 TF patients and from 14 patients with ventricular septal defects (VSDs), used as a comparison group. The Notch pathway was analyzed by estimating the expression of Notch-related genes by qPCR. Differentiation and proliferation capacity of the cells was estimated. RESULTS: The TF-derived cells demonstrated a dysregulated pattern of Notch-related gene expression comparing to VSD-derived cells. Correlation of Notch signaling activation level by HEY1/HES1 expression level with proliferation and cardiogenic-like differentiation of cardiac mesenchymal cells was observed but not with clinical parameters nor with the age of the patients. CONCLUSIONS: The data suggest a contribution of dysregulated Notch signaling to the pathogenesis of tetralogy of Fallot and importance of Notch signaling level for the functional state of cardiac mesenchymal cells, which could be critical considering these cells for potential cell therapy approaches.

Proteomic Profiling of the Human Fetal Multipotent Mesenchymal Stromal Cells Secretome.

Secretome of multipotent mesenchymal stromal cells (MSCs) is actively used in biomedical applications such as alveolar bone regeneration, treatment of cardiovascular disease, and neurodegenerative disorders. Nevertheless, hMSCs have low proliferative potential and production of the industrial quantity of their secretome might be challenging. Human fetal multipotent mesenchymal stromal cells (FetMSCs) isolated from early human embryo bone marrow are easy to expand and might be a potential source for pharmaceutical substances production based on their secretome. However, the secretome of FetMSCs was not previously analyzed. Here, we describe the secretome of FetMSCs using LC-MALDI shotgun proteomics. We identified 236 proteins. Functional annotation of the identified proteins revealed their involvement in angiogenesis, ossification, regulation of apoptosis, and immune response processes, which made it promising for biomedical applications. The proteins identified in the FetMSCs secretome are involved in the same biological processes as proteins from previously described adult hMSCs secretomes. Nevertheless, many of the common hMSCs secretome components (such as VEGF, FGF, Wnt and TGF-ß) have not been identified in the FetMSCs secretome.

Human aortic endothelial cells have osteogenic Notch-dependent properties in co-culture with aortic smooth muscle cells.

Cardiovascular calcification is one of the leading reasons of morbidity and mortality in Western countries and has many similarities to osteogenesis. The role of smooth muscle calcific transformation is well established for atherogenic lesions, but mechanisms driving initial stages of proosteogenic cell fate commitment in big vessels remain poorly understood. The role of endothelial and underlying interstitial cell interaction in driving cellular decisions is emerging from recent studies. The aim of this study was to analyze co-culture of endothelial and smooth muscle cells in vitro in acquiring proosteogenic phenotype. We co-cultured human aortic endothelial cells (EC) and human aortic smooth muscle cells (SMC) and analyzed osteogenic phenotype by ALP staining and proosteogenic gene expression by qPCR in co-cultures and in separate cellular types after magnetic CD31-sorting. In EC and SMC co-cultures osteogenic phenotype was induced as well as activated expression of RUNX2, POSTIN, BMP2/4, SOX5, COL1A SMC; co-culture of EC with SMC induced NOTCH1, NOTCH3, NOTCH4 and HEY1 expression; Notch activation by lentiviral activated Notch intracellular domain induced expression of RUNX2, OPN, POSTIN in SMC; NOTCH1 and NOTCH3 and HEY1 were selectively induced in EC during co-culture. We conclude that endothelial cells are capable of driving smooth muscle calcification via cell-cell contact and activation of Notch signaling.

Notch-dependent EMT is attenuated in patients with aortic aneurysm and bicuspid aortic valve.

Bicuspid aortic valve is the most common congenital heart malformation and the reasons for the aortopathies associated with bicuspid aortic valve remain unclear. NOTCH1 mutations are associated with bicuspid aortic valve and have been found in individuals with various left ventricular outflow tract abnormalities. Notch is a key signaling during cardiac valve formation that promotes the endothelial-to-mesenchymal transition. We address the role of Notch signaling in human aortic endothelial cells from patients with bicuspid aortic valve and aortic aneurysm. Aortic endothelial cells were isolated from tissue fragments of bicuspid aortic valve-associated thoracic aortic aneurysm patients and from healthy donors. Endothelial-to-mesenchymal transition was induced by activation of Notch signaling. Effectiveness of the transition was estimated by loss of endothelial and gain of mesenchymal markers by immunocytochemistry and qPCR. We show that aortic endothelial cells from the patients with aortic aneurysm and bicuspid aortic valve have down regulated Notch signaling and fail to activate Notch-dependent endothelial-to-mesenchymal transition in response to its stimulation by different Notch ligands. Our findings support the idea that bicuspid aortic valve and associated aortic aneurysm is associated with dysregulation of the entire Notch signaling pathway independently on the specific gene mutation.

Various lamin A/C mutations alter expression profile of mesenchymal stem cells in mutation specific manner.

Various mutations in LMNA gene, encoding for nuclear lamin A/C protein, lead to laminopathies and contribute to over ten human disorders, mostly affecting tissues of mesenchymal origin such as fat tissue, muscle tissue, and bones. Recently it was demonstrated that lamins not only play a structural role providing communication between extra-nuclear structures and components of cell nucleus but also control cell fate and differentiation. In our study we assessed the effect of various LMNA mutations on the expression profile of mesenchymal multipotent stem cells (MMSC) during adipogenic and osteogenic differentiation. We used lentiviral approach to modify human MMSC with LMNA-constructs bearing mutations associated with different laminopathies--G465D, R482L, G232E, R527C, and R471C. The impact of various mutations on MMSC differentiation properties and expression profile was assessed by colony-forming unit analysis, histological staining, expression of the key differentiation markers promoting adipogenesis and osteogenesis followed by the analysis of the whole set of genes involved in lineage-specific differentiation using PCR expression arrays. We demonstrate that various LMNA mutations influence the differentiation efficacy of MMSC in mutation-specific manner. Each LMNA mutation promotes a unique expression pattern of genes involved in a lineage-specific differentiation and this pattern is shared by the phenotype-specific mutations.

The Role of NOTCH Pathway Genes in the Inherited Susceptibility to Aortic Stenosis.

The NOTCH-signaling pathway is responsible for intercellular interactions and cell fate commitment. Recently, NOTCH pathway genes were demonstrated to play an important role in aortic valve development, leading to an increased calcified aortic valve disease (CAVD) later in life. Here, we further investigate the association between genetic variants in the NOTCH pathway genes and aortic stenosis in a case-control study of 90 CAVD cases and 4723 controls using target panel sequencing of full-length 20 genes from a NOTCH-related pathway (DVL2, DTX2, MFNG, NUMBL, LFNG, DVL1, DTX4, APH1A, DTX1, APH1B, NOTCH1, ADAM17, DVL3, NCSTN, DTX3L, ILK, RFNG, DTX3, NOTCH4, PSENEN). We identified a common intronic variant in NOTCH1, protecting against CAVD development (rs3812603), as well as several rare and unique new variants in NOTCH-pathway genes (DTX4, NOTCH1, DTX1, DVL2, NOTCH1, DTX3L, DVL3), with a prominent effect of the protein structure and function.

Interplay between BMP2 and Notch signaling in endothelial-mesenchymal transition: implications for cardiac fibrosis.

Background: The endothelial-to-mesenchymal transition (EndoMT) is a crucial process in cardiovascular development and disorders. Cardiac fibrosis, characterized by excessive collagen deposition, occurs in heart failure, leading to the organ remodeling. Embryonic signaling pathways such as bone morphogenetic protein 2 (BMP2) and Notch are involved in its regulation. However, the interplay between these pathways in EndoMT remains unclear. Methods: This study investigates the downstream targets of Notch and BMP2 and their effect on EndoMT markers in cardiac mesenchymal cells (CMCs) and human umbilical vein endothelial cells (HUVECs). We transduced cell cultures with vectors carrying intracellular domain of NOTCH1 (NICD) and/or BMP2 and evaluated gene expression and activation of EndoMT markers. Results: The results suggest that the Notch and BMP2 signaling pathways have common downstream targets that regulate EndoMT. The activation of BMP2 and Notch is highly dependent on cell type, and co-cultivation of CMCs and HUVECs produced opposing cellular responses to target gene expression and α-smooth muscle actin (α-SMA) synthesis. Conclusions: The balance between Notch and BMP2 signaling determines the outcome of EndoMT and fibrosis in the heart. The study's findings highlight the need for further research to understand the interaction between Notch and BMP2 in the heart and develop new therapeutic strategies for treating cardiac fibrosis.

Purinergic Signaling in Pathologic Osteogenic Differentiation of Aortic Valve Interstitial Cells from Patients with Aortic Valve Calcification.

Purinergic signaling is associated with a vast spectrum of physiological processes, including cardiovascular system function and, in particular, its pathological calcifications, such as aortic valve stenosis. Aortic valve stenosis (AS) is a degenerative disease for which there is no cure other than surgical replacement of the affected valve. Purinergic signaling is known to be involved in the pathologic osteogenic differentiation of valve interstitial cells (VIC) into osteoblast-like cells, which underlies the pathogenesis of AS. ATP, its metabolites and related nucleotides also act as signaling molecules in normal osteogenic differentiation, which is observed in pro-osteoblasts and leads to bone tissue development. We show that stenotic and non-stenotic valve interstitial cells significantly differ from each other, especially under osteogenic stimuli. In osteogenic conditions, the expression of the ecto-nucleotidases ENTPD1 and ENPP1, as well as ADORA2b, is increased in AS VICs compared to normal VICs. In addition, AS VICs after osteogenic stimulation look more similar to osteoblasts than non-stenotic VICs in terms of purinergic signaling, which suggests the stronger osteogenic differentiation potential of AS VICs. Thus, purinergic signaling is impaired in stenotic aortic valves and might be used as a potential target in the search for an anti-calcification therapy.

Analysis of Prevalence and Clinical Features of Aortic Stenosis in Patients with and without Bicuspid Aortic Valve Using Machine Learning Methods.

Aortic stenosis (AS) is the most commonly diagnosed valvular heart disease, and its prevalence increases with the aging of the general population. However, AS is often diagnosed at a severe stage, necessitating surgical treatment, due to its long asymptomatic period. The objective of this study was to analyze the frequency of AS in a population of cardiovascular patients using echocardiography (ECHO) and to identify clinical factors and features associated with these patient groups. We utilized machine learning methods to analyze 84,851 echocardiograms performed between 2010 and 2018 at the National Medical Research Center named after V.A. Almazov. The primary indications for ECHO were coronary artery disease (CAD) and hypertension (HP), accounting for 33.5% and 14.2% of the cases, respectively. The frequency of AS was found to be 13.26% among the patients (n = 11,252). Within our study, 1544 patients had a bicuspid aortic valve (BAV), while 83,316 patients had a tricuspid aortic valve (TAV). BAV patients were observed to be younger compared to TAV patients. AS was more prevalent in the BAV group (59%) compared to the TAV group (12%), with a p-value of <0.0001. By employing a machine learning algorithm, we randomly identified significant features present in AS patients, including age, hypertension (HP), aortic regurgitation (AR), ascending aortic dilatation (AscAD), and BAV. These findings could serve as additional indications for earlier observation and more frequent ECHO in specific patient groups for the earlier detection of developing AS.

Streptococcal arginine deiminase regulates endothelial inflammation, mTOR pathway and autophagy.

Endothelial cells (EC) are active participants in the inflammation process. During the infection, the change in endothelium properties provides the leukocyte infiltrate formation and restrains pathogen dissemination due to coagulation control. Pathogenic microbes are able to change the endothelium properties and functions in order to invade the bloodstream and disseminate in the host organism. Arginine deiminase (ADI), a bacterial arginine-hydrolyzing enzyme, which causes the amino acid deficiency, important for endothelium biology. Previous research implicates altered metabolism of arginine in the development of endothelial dysfunction and inflammation. It was shown that arginine deficiency, as well as overabundance affects the balance of mechanical target of rapamycin (mTOR)/S6 kinase (S6K) pathway, arginase and endothelial nitric oxide synthase (eNOS) resulted in reactive oxygen species (ROS) production and EC activation. ADI creating a deficiency of arginine can interfere cellular arginine-dependent processes. Thus, this study was aimed at investigation of the influence of streptococcal ADI on the metabolism and inflammations of human umbilical vein endothelial cells (HUVEC). The action of ADI was studied by comparing the effect Streptococcus pyogenes M49-16 paternal strain expressing ADI and its isogenic mutant M49-16delArcA with the inactivated gene ArcA. Based on comparison of the parental and mutant strain effects, it can be concluded, that ADI suppressed mTOR signaling pathway and enhanced autophagy. The processes failed to return to the basic level with arginine supplement. Our study also demonstrates that ADI suppressed endothelial proliferation, disrupted actin cytoskeleton structure, increased phospho-NF-κB p65, CD62P, CD106, CD54, CD142 inflammatory molecules expression, IL-6 production and lymphocytes-endothelial adhesion. In spite of the ADI-mediated decrease in arginine concentration in the cell-conditioned medium, the enzyme enhanced the production of nitric oxide in endothelial cells. Arginine supplementation rescued proliferation, actin cytoskeleton structure, brought NO production to baseline and prevented EC activation. Additional evidence for the important role of arginine bioavailability in the EC biology was obtained. The results allow us to consider bacterial ADI as a pathogenicity factor that can potentially affect the functions of endothelium.

Antibacterial Activity and Cytocompatibility of Electrospun PLGA Scaffolds Surface-Modified by Pulsed DC Magnetron Co-Sputtering of Copper and Titanium.

Biocompatible poly(lactide-co-glycolide) scaffolds fabricated via electrospinning are having promising properties as implants for the regeneration of fast-growing tissues, which are able to degrade in the body. The hereby-presented research work investigates the surface modification of these scaffolds in order to improve antibacterial properties of this type of scaffolds, as it can increase their application possibilities in medicine. Therefore, the scaffolds were surface-modified by means of pulsed direct current magnetron co-sputtering of copper and titanium targets in an inert atmosphere of argon. In order to obtain different amounts of copper and titanium in the resulting coatings, three different surface-modified scaffold samples were produced by changing the magnetron sputtering process parameters. The success of the antibacterial properties' improvement was tested with the methicillin-resistant bacterium Staphylococcus aureus. In addition, the resulting cell toxicity of the surface modification by copper and titanium was examined using mouse embryonic and human gingival fibroblasts. As a result, the scaffold samples surface-modified with the highest copper to titanium ratio show the best antibacterial properties and no toxicity against mouse fibroblasts, but have a toxic effect to human gingival fibroblasts. The scaffold samples with the lowest copper to titanium ratio display no antibacterial effect and toxicity. The optimal poly(lactide-co-glycolide) scaffold sample is surface-modified with a medium ratio of copper and titanium that has antibacterial properties and is non-toxic to both cell cultures.

LMNA mutation leads to cardiac sodium channel dysfunction in the Emery-Dreifuss muscular dystrophy patient.

Pathogenic variants in the LMNA gene are known to cause laminopathies, a broad range of disorders with different clinical phenotypes. LMNA genetic variants lead to tissue-specific pathologies affecting various tissues and organs. Common manifestations of laminopathies include cardiovascular system abnormalities, in particular, cardiomyopathies and conduction disorders. In the present study, we used induced pluripotent stem cells from a patient carrying LMNA p.R249Q genetic variant to create an in vitro cardiac model of laminopathy. Induced pluripotent stem cell-derived cardiomyocytes with LMNA p.R249Q genetic variant showed a decreased sodium current density and an impaired sodium current kinetics alongside with changes in transcription levels of cardiac-specific genes. Thus, we obtained compelling in vitro evidence of an association between LMNA p.R249Q genetic variant and cardiac-related abnormalities.

Redo aortic valve-sparing root replacement for failing autograft after the Ross procedure: A case report.

INTRODUCTION AND IMPORTANCE: Late pulmonary autograft dilatation is observed in 10-20 % of patients after the Ross procedure, more often during the second decade of follow-up. Composite aortic root replacement with a valved conduit is the most common redo procedure. An aortic valve-sparing root replacement does not require lifelong anticoagulation and may significantly decrease the risk of complications, associated with a valve prosthesis. PRESENTATION OF THE CASE: We report a case of late pulmonary autograft dilatation developed after the Ross procedure. The annual transthoracic echocardiography after 20 years revealed severe dilatation of the pulmonary autograft, measuring 60 mm in diameter. The patient underwent a successful elective redo valve-sparing aortic root replacement (David I procedure). The aortic cross-clamp time was 144 min, and the CPB time was 181 min. The patient had an uneventful midterm postoperative course. CLINICAL DISCUSSION: Late pulmonary autograft failure after the Ross procedure is a relatively rare condition, leading to repeat operation. Late autograft failure can contribute to aortic regurgitation, heart failure, and death due to ascending aortic dissection and rupture. Several research groups reported good early and midterm results of redo valve-sparing root replacement in such cases. In a reoperative valve-sparing root replacement after the Ross procedure, the portion of the native aorta with the adjacent part of the autograft may complicate the aortic root proper sizing. CONCLUSION: Redo valve-sparing root replacement (David I procedure) is a viable option in pulmonary autograft dilatation with unaffected valve leaflets.