Bicuspid aortic valve-associated aortopathy unraveling the molecular signature

Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysm compared with individuals with a tricuspid aortic valve (TAV). A crucial factor involved in vascular remodeling during aneurysm development is transforming growth factor - β (TGF - β) and impaired signaling of this pathway can alter important extracellular matrix (ECM) protein such us fibronectin and collagen and thereby explaining the increased aneurysm susceptibility of BAV patients. The overall aim of this thesis was to investigate the BAV - associated aortopathy in relation to the TGF - β signaling pathway. A lternatively spliced extra domain A (EDA) of fibronectin (FN) has an essential role in tissue repair . In paper I, t he mR NA expression of FN splice forms was analyzed by Affymetrix Exon arrays in dilated and non - dilated ascending aorta of TAV (40 individuals) and BAV patients (69 individuals). EDA - FN was significantly increased only in TAV dilated aortas. Upon TGF - β treatmen t, vascular smooth muscle cells (vSMCs) isolated from TAV aortas were able to enhance the formation of EDA - FN whereas cells derived from BAV patients could not influence fibronectin splicing. Multivariate and univariate data analyses of mRNA expression sug gested that differences in the TGF - β signaling pathway may explain the impaired EDA inclusion in BAV patients. In paper II , m ultivariate techniques were applied to all exons (n = 614) of the TGF - β pathway in order to analyze alternative splicing in the TGF - β pathway. Alternative splicing mechanisms were found to be important in the development of aneurysm in both BAV and TAV patients. Furthermore, the pattern of alternative splicing is clearly different between TAV and BAV patients. Differential splicing wa s specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicing of 61 exons were shared between the two phenotypes. This suggested that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGF - β pathw ay. In paper III, c ollagen homeostasis in non - dilated and dilated aorta s of BAV patients was studied and compared to non - dilated and dilated aortas taken from tricuspid aortic valve patients as reference. Ascending aortas from 56 patients were used for bio chemical and morphological analyses of collagen. Collagen turnover rates were similar in non - dilated and dilated aortas of BAV patients, showing that aneurysm formation in BAV is, in contrast to TAV, not associated with an increased collagen turnover. In a ddition, the ratio of hydroxylysyl pyridinoline (HP) to lysyl pyridinoline (LP), two distinct forms of collagen cross - linking, was lower in dilated aortas from patients with BAV, which hints at a defect in the posttranslational collagen modification associ ated with BAV. In paper IV , t he response to TGF - β was analyzed in primary aortic smooth muscle cells isolated from 7 BAV and 5 BAV patients and 217 genes were found differentially expressed following TGF - β1 treatment in BAV vSMCs, whereas no gene was sign ificantly altered between treated/untreated vSMCs of TAV patients. Majority of genes were down - regulated and enriched in genes involved in angiogenesis and formation of focal adhesion. Importantly, principle component analysis based on the 217 genes demons trated that there was a clear difference in expression of these genes in intima/media region of dilated ascending aorta of BAV and TAV patients. In conclusion, the understanding of impairments in the TGF - β signaling pathway could be the key to unravel the molecular mechanism s underlying BAV - associated aortopathy

Allo Beta Cell transplantation: specific features, unanswered questions, and immunological challenge

Type 1 diabetes (T1D) presents a persistent medical challenge, demanding innovative strategies for sustained glycemic control and enhanced patient well-being. Beta cells are specialized cells in the pancreas that produce insulin, a hormone that regulates blood sugar levels. When beta cells are damaged or destroyed, insulin production decreases, which leads to T1D. Allo Beta Cell Transplantation has emerged as a promising therapeutic avenue, with the goal of reinstating glucose regulation and insulin production in T1D patients. However, the path to success in this approach is fraught with complex immunological hurdles that demand rigorous exploration and resolution for enduring therapeutic efficacy. This exploration focuses on the distinct immunological characteristics inherent to Allo Beta Cell Transplantation. An understanding of these unique challenges is pivotal for the development of effective therapeutic interventions. The critical role of glucose regulation and insulin in immune activation is emphasized, with an emphasis on the intricate interplay between beta cells and immune cells. The transplantation site, particularly the liver, is examined in depth, highlighting its relevance in the context of complex immunological issues. Scrutiny extends to recipient and donor matching, including the utilization of multiple islet donors, while also considering the potential risk of autoimmune recurrence. Moreover, unanswered questions and persistent gaps in knowledge within the field are identified. These include the absence of robust evidence supporting immunosuppression treatments, the need for reliable methods to assess rejection and treatment protocols, the lack of validated biomarkers for monitoring beta cell loss, and the imperative need for improved beta cell imaging techniques. In addition, attention is drawn to emerging directions and transformative strategies in the field. This encompasses alternative immunosuppressive regimens and calcineurin-free immunoprotocols, as well as a reevaluation of induction therapy and recipient preconditioning methods. Innovative approaches targeting autoimmune recurrence, such as CAR Tregs and TCR Tregs, are explored, along with the potential of stem stealth cells, tissue engineering, and encapsulation to overcome the risk of graft rejection. In summary, this review provides a comprehensive overview of the inherent immunological obstacles associated with Allo Beta Cell Transplantation. It offers valuable insights into emerging strategies and directions that hold great promise for advancing the field and ultimately improving outcomes for individuals living with diabetes

Outcomes of pregnancies after kidney transplantation: lessons learned from CKD. A comparison of transplanted, nontransplanted chronic kidney disease patients and low-risk pregnancies: a multicenter nationwide analysis.

BACKGROUND: Kidney transplantation (KT) may restore fertility in CKD. The reasons why materno-foetal outcomes are still inferior to the overall population are only partially known. Comparison with the CKD population may offer some useful insights for management and counselling.Aim of this study was to analyse the outcomes of pregnancy after KT, compared with a large population of non-transplanted CKD patients and with low-risk control pregnancies, observed in Italy the new millennium. METHODS: We selected 121 live-born singletons after KT (Italian study group of kidney in pregnancy, national coverage about 75%), 610 live-born singletons in CKD and 1418 low-risk controls recruited in 2 large Italian Units, in the same period (2000-2014). The following outcomes were considered: maternal and foetal death; malformations; preterm delivery; small for gestational age baby (SGA); need for the neonatal intensive care unit (NICU); doubling of serum creatinine or increase in CKD stage. Data were analysed according to kidney diseases, renal function (staging according to CKD-EPI), hypertension, maternal age, partity, ethnicity. RESULTS: Materno-foetal outcomes are less favourable in CKD and KT as compared with the low-risk population. CKD stage and hypertension are important determinants of results. KT patients with e-GFR >90 have worse outcomes compared with CKD stage 1 patients; the differences level off when only CKD patients affected by glomerulonephritis or systemic diseases ('progressive CKD') are compared with KT. In the multivariate analysis, risk for preterm and early-preterm delivery was linked to CKD stage (2-5 versus 1: RR 3.42 and 3.78) and hypertension (RR 3.68 and 3.16) while no difference was associated with being a KT or a CKD patient. CONCLUSIONS: The materno-foetal outcomes in patients with kidney transplantation are comparable with those of nontransplanted CKD patients with similar levels of kidney function impairment and progressive and/or immunologic kidney diseas

Organ-on-a-chip technology : a novel approach to investigate cardiovascular diseases

The development of organs-on-chip (OoC) has revolutionized in vitro cell-culture experiments by allowing a better mimicry of human physiology and pathophysiology that has consequently led researchers to gain more meaningful insights into disease mechanisms. Several models of hearts-on-chips and vessels-on-chips have been demonstrated to recapitulate fundamental aspects of the human cardiovascular system in the recent past. These 2D and 3D systems include synchronized beating cardiomyocytes in hearts-on-chips and vessels-on-chips with layer-based structures and the inclusion of physiological and pathological shear stress conditions. The opportunities to discover novel targets and to perform drug testing with chip-based platforms have substantially enhanced, thanks to the utilization of patient-derived cells and precise control of their microenvironment. These organ models will provide an important asset for future approaches to personalized cardiovascular medicine and improved patient care. However, certain technical and biological challenges remain, making the global utilization of OoCs to tackle unanswered questions in cardiovascular science still rather challenging. This review article aims to introduce and summarize published work on hearts- and vessels-on chips but also to provide an outlook and perspective on how these advanced in vitro systems can be used to tailor disease models with patient-specific characteristics.De tre sista författarna delar sistaförfattarskapet</p

Impaired Collagen Biosynthesis and Cross-linking in Aorta of Patients With Bicuspid Aortic Valve

<p>Background-Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysm. In the present study, collagen homeostasis in nondilated and dilated aorta segments from patients with BAV was studied, with normal and dilated aortas from tricuspid aortic valve (TAV) patients as reference.</p><p>Methods and Results-Ascending aortas from 56 patients were used for biochemical and morphological analyses of collagen. mRNA expression was analyzed in 109 patients. Collagen turnover rates were similar in nondilated and dilated aortas of BAV patients, showing that aneurysmal formation in BAV is, in contrast to TAV, not associated with an increased collagen turnover. However, BAV in general was associated with an increased aortic collagen turnover compared with nondilated aortas of TAV patients. Importantly, the ratio of hydroxylysyl pyridinoline (HP) to lysyl pyridinoline (LP), 2 distinct forms of collagen cross-linking, was lower in dilated aortas from patients with BAV, which suggests that BAV is associated with a defect in the posttranslational collagen modification. This suggests a deficiency at the level of lysyl hydroxylase (PLOD1), which was confirmed by mRNA and protein analyses that showed reduced PLOD1 expression but normal lysyl oxidase expression in dilated aortas from patients with BAV. This suggests that impaired collagen cross-linking in BAV patients may be attributed to changes in the expression and/or activity of PLOD1.</p><p>Conclusions-Our results demonstrate an impaired biosynthesis and posttranslational modification of collagen in aortas of patients with BAV, which may explain the increased aortic aneurysm formation in BAV patients.</p>

Changes in Endocan and Dermatan Sulfate Are Associated with Biomechanical Properties of Abdominal Aortic Wall during Aneurysm Expansion and Rupture

Background and aims: The study aimed to assess the potential of proteoglycans (PGs) and collagens as serological biomarkers in the abdominal aortic aneurysm (AAA). Furthermore, we investigated the underlying mechano-biological interactions and signaling pathways. Methods: Tissue and serum samples from patients with ruptured AAA (rAAA; n = 29), elective AAA (eAAA; n = 78), and healthy individuals (n = 8) were evaluated by histology, immunohistochemistry, and enzyme-linked immunosorbent assay, and mechanical properties were assessed by tensile tests. Regulatory pathways were determined by membrane-based sandwich immunoassay. Results: In AAA samples, collagen type I and III (Col1 and Col3), chondroitin sulfate, and dermatan sulfate (DS) were significantly increased compared with controls (3.0-, 3.2-, 1.3-, and 53-fold; p < 0.01). Col1 and endocan were also elevated in the serum of AAA patients (3.6- and 6.0-fold; p < 0.01), while DS was significantly decreased (2.5-fold; p < 0.01). Histological scoring showed increased total PGs and focal accumulation in rAAA compared with eAAA. Tissue β-stiffness was higher in rAAA compared with eAAA (2.0-fold, p = 0.02). Serum Col1 correlated with maximum tensile force and failure tension (r = 0.448 and 0.333; p < 0.01, and r = 0.02), tissue endocan correlated with α-stiffness (r = 0.340; p < 0.01). Signaling pathways in AAA were associated with extracellular matrix synthesis and vascular smooth muscle cell proliferation. In particular, Src family kinases and platelet-derived growth factor- and epidermal growth factor-related proteins seem to be involved. Conclusion: Our findings reveal a structural association between collagen and PGs and their response to changes in mechanical loads in AAA. Particularly Col1 and endocan reflect the mechano-biological conditions of the aortic wall also in the patient's serum and might serve for AAA risk stratification

Diverging Alternative Splicing Fingerprints in the Transforming Growth Factor-β Signaling Pathway Identified in Thoracic Aortic Aneurysms

Impaired regulation of the transforming growth factor-β (TGFβ) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGFβ pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGFβ pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGFβ pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcription–polymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGFβ pathway

The circular RNA Ataxia Telangiectasia Mutated regulates oxidative stress in smooth muscle cells in expanding abdominal aortic aneurysms

An abdominal aortic aneurysm (AAA) is a pathological widening of the aortic wall characterized by loss of smooth muscle cells (SMCs), extracellular matrix degradation, and local inflammation. This condition is often asymptomatic until rupture occurs, leading to high morbidity and mortality rates. Diagnosis is mostly accidental and the only currently available treatment option remains surgical intervention. Circular RNAs (circRNAs) represent a novel class of regulatory non-coding RNAs that originate from backsplicing. Their highly stable loop structure, combined with a remarkable enrichment in body fluids, make circRNAs promising disease biomarkers. We investigated the contribution of circRNAs to AAA pathogenesis and their potential application to improve AAA diagnostics. Gene expression analysis revealed the presence of deregulated circular transcripts stemming from AAA-relevant gene loci. Among these, the circRNA to the Ataxia Telangiectasia Mutated gene (cATM) was upregulated in human AAA specimens, in AAA-derived SMCs, and serum samples collected from aneurysm patients. In primary aortic SMCs, cATM increased upon angiotensin II and doxorubicin stimulation, while its silencing triggered apoptosis. Higher cATM levels made AAA-derived SMCs less vulnerable to oxidative stress, compared with control SMCs. These data suggest that cATM contributes to elicit an adaptive oxidative-stress response in SMCs and provides a reliable AAA disease signature