Heparan Sulfate Proteoglycans (HSPGs) are important cell surface and Extracellular Matrix (ECM) maestros involved in the orchestration of multiple cellular events in physiology and pathology. These glycoconjugates bind to various bioactive proteins via their Heparan Sulfate (HS) chains, but also through the protein backbone, and function as scaffolds for protein-protein interactions, modulating extracellular ligand gradients, cell signalling networks and cell-cell/cell-ECM interactions. The structural features of HS chains, including length and sulfation patterns, are crucial for the biological roles displayed by HSPGs, as these features determine HS chains binding affinities and selectivity. The large HS structural diversity results from a tightly controlled biosynthetic pathway that is differently regulated in different organs, stages of development and pathologies, including cancer. This review addresses the regulatory mechanisms underlying HS biosynthesis, with a particular focus on the catalytic activity of the enzymes responsible for HS glycan sequences and sulfation motifs, namely D-Glucuronyl C5-Epimerase, N- and O-Sulfotransferases. Moreover, we provide insights on the impact of different HS structural epitopes over HSPG-protein interactions and cell signalling, as well as on the effects of deregulated expression of HS modifying enzymes in the development and progression of cancer. Finally, we discuss the clinical potential of HS biosynthetic enzymes as novel targets for therapy, and highlight the importance of developing new HS-based tools for better patients’ stratification and cancer treatment.

In an era when cancer glycobiology research is exponentially growing, we are witnessing a progressive translation of the major scientific findings to the clinical practice with the overarching aim of improving cancer patients’ management. Many mechanistic cell biology studies have demonstrated that heparan sulfate (HS) glycosaminoglycans are key molecules responsible for several molecular and biochemical processes, impacting extracellular matrix properties and cellular functions. HS can interact with a myriad of different ligands, and therefore, hold a pleiotropic role in regulating the activity of important cellular receptors and downstream signalling pathways. The aberrant expression of HS glycan chains in tumours determines main malignant features, such as cancer cell proliferation, angiogenesis, invasion and metastasis. In this review, we devote particular attention to HS biological activities, its expression profile and modulation in cancer. Moreover, we highlight HS clinical potential to improve both diagnosis and prognosis of cancer, either as HS-based biomarkers or as therapeutic targets.

Imprinting errors have been described in spermatozoa from infertile patients with oligozoospermia and azoospermia. However, little is known about methylation of imprinted genes in other spermatogenic cells from azoospermic patients. Therefore, we aimed to evaluate the methylation status of single CpGs located in the differentially methylated regions (DMRs) of two imprinted genes, one paternally (H19) and one maternally (MEST) methylated, in primary spermatocytes of azoospermic patients presenting complete (MAc, n = 7) and incomplete (MAi, n = 8) maturation arrest, as well as in other spermatogenic cells from MAi patients that presented focus of complete spermatogenesis in some seminiferous tubules. We observed H19 imprinting errors in primary spermatocytes from one MAi patient and MEST imprinting errors in one MAi and two MAc patients. Additionally, H19 imprinting errors were observed in elongated spermatids/spermatozoa from one MAi patient. Nevertheless, no statistical differences were found for H19 and MEST global methylation levels (percentage of methylated and unmethylated CpGs, respectively) between patients with complete and incomplete MA and also between MA groups and a control group. These results provide further evidence that imprinting errors occur in spermatogenic cells from patients presenting impaired spermatogenesis, as we and others have previously described in ejaculated and testicular spermatozoa. As paternal imprinting errors can be transmitted to the embryo by the sperm cell, they can provide a possible explanation for poor embryo development and/or low pregnancy rates as correct expression of imprinted genes is crucial for embryo and placental development and function. Therefore, in cases with male factor infertility where unsuccessful in vitro fertilization (IVF) treatments are recurrent, analysis of imprinting marks in spermatozoa might be a useful diagnostic tool.

The placenta is a temporary organ that is essential for supporting mammalian embryo and fetal development. Understanding the molecular mechanisms underlying trophoblast differentiation and placental function may contribute to improving the diagnosis and treatment of obstetric complications. Epigenetics plays a significant role in the regulation of gene expression, particularly at imprinted genes, which are fundamental in the control of placental development. The Ten-Eleven-Translocation enzymes are part of the epigenetic machinery, converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). DNA hydroxymethylation is thought to act as an intermediate in the DNA demethylation mechanism and potentially be a stable and functionally relevant epigenetic mark on its own. The role of DNA hydroxymethylation during differentiation and development of the placenta is not fully understood but increasing knowledge in this field will help to evaluate its potential role in pregnancy complications. This review focuses on DNA hydroxymethylation and its epigenetic regulators in human and mouse placental development and function. Additionally, we address 5hmC in the context of genomic imprinting mechanism and in pregnancy complications, such as intrauterine growth restriction, preeclampsia and pregnancy loss. The cumulative findings show that DNA hydroxymethylation might be important for the control of gene expression in the placenta and suggest a dynamic role in the differentiation of trophoblast cell types during gestation.

COVID-19 pandemic has unquestionably influenced care of acute myocardial infarction (AMI). Still, its impact on patients (pts) characteristics, presentation, treatment, and outcomes remains not well established in late pandemic times. To address this issue, we performed a prospective study of type-1 AMI pts admitted in a tertiary care hospital. Pts were enrolled during 6-months in 2019 (n=122; pre-COVID-19 (PC) group) and in 2021 (n=196; late-COVID-19 (C) group). Data was based on pts interview and review of medical records. Age and gender distribution, as well as ST/Non-ST-Elevation Myocardial Infarction (STEMI/NSTEMI) proportion and access to coronariography and revascularization were similar between groups. C pts presented more pre-existing established cardiovascular disease (CVD) (43% vs 30%; p=0,03); more frequent description of typical chest pain (94% vs 84%; p=0,002); higher levels of pain intensity, in a 0-10 scale (8±2 vs 7±2; p=0,02); higher frequencies of AMI complications (27% vs 15%; p=0,01) and worse Killip (K) class evolution (K≥2 in 22% C vs 13% PC pts; p=0,05). In conclusion, late pandemic AMI pts presented worse in-hospital outcomes in our study, though pre-hospital and hospital care were comparable to pre-pandemic times. Covid pts had a higher burden of pre-existing established CVD and a more typical and intense symptom presentation. Therefore, it can be hypothesized that "sicker" pts continued to look for help when presenting AMI symptoms, while "less sick" pts and the ones with less typical and intense symptoms possibly avoided contact with health care services during late pandemic period.

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A 69-year-old male presented with typical angina while showering. He had history of CABG in 2008 (left internal mammary arterial [LIMA] to the first marginal and intermediate arteries and RIMA to the LAD artery), with preserved biventricular systolic function. On physical examination, an upper-arm systolic blood pressure differential >20mmHg and a decreased pulse amplitude on the left side was found. ECG revealed sinus tachycardia with RBBB, ST-segment depression and inverted T-waves in the lateral and inferior leads. Troponin and BNP levels were elevated. Echocardiogram showed reduced left ventricular ejection fraction (22%) and de novo akinesia of the inferior and posterior walls. The diagnosis of non-ST-segment elevation myocardial infarction was assumed. Coronary angiography revealed patent bypass grafts without disease and a 90% stenosis of the left subclavian artery (LSA) proximal to the ostia of the LIMA, with retrograde flow ‘stealing’ the myocardial blood supply. Ultrasound scan detected systolic reversal of flow in the left vertebral artery, suggesting subclavian-vertebral steal phenomenon. CT-angiography revealed a 14-mm stenosis with a useful lumen of 2 mm in the LSA. A percutaneous balloon angioplasty with stenting of the LSA was performed by the Vascular team, restoring the normal blood supply. Coronary subclavian steal syndrome can manifest as myocardial infarction or heart failure, due to functional LIMA graft failure by inadequate blood supply to the myocardium. Anamnesis and physical examination are fundamental in order not to miss the diagnosis. Subclavian angiography is the gold standard to confirm the diagnosis and can be performed during coronary angiography. Revascularization of the LSA is the definitive treatment. Figure 1Coronary angiography revealed chronic occluded native coronary vessels with patency and no significant disease of the bypass grafts, and high grade (90%) left subclavian artery (LSA) stenosis proximal to the ostia of the LIMA, conditioning the blood flow to the left upper limb and ‘stealing’ the myocardial blood supply because of retrograde flow in the LIMA graft.

Heparan Sulfate ProteoGlycans (HSPGs) are essential components of cells’ glycocalyx and Extracellular Matrix (ECM), which play key roles in cell physiology and in pathological scenarios. By binding to multiple biological ligands, via HS glycosaminoglycan (GAG) chains, HSPGs modulate cancer cells’ interaction with ECM and signalling networks, ultimately controlling tumour microenvironment and disease development. The aim of this work was to disclose the regulatory mechanisms underlying HSPGs biosynthetic pathways in gastric cancer context. Furthermore, we evaluated the impact of cellular GAGs content in cancer cell signalling and motility features. We developed glycoengineered gastric cancer cell models lacking specific glycosyltransferases involved in HS biosynthesis, Exostosin Like Glycosyltransferase 2 (EXTL2) and EXTL3. Biochemical characterization revealed that abrogation of EXTL2 or EXTL3 impacts both HS and Chondroitin Sulfate (CS) cell levels, as well as GAGs structural conformations, supporting that both enzymes exert broad regulatory roles in GAGs biosynthesis pathways. We observed that while EXTL3 is key for initiating the synthesis of HS chains, in detriment of CS biosynthesis, EXTL2 functions as a negative regulator of HS biosynthesis. Gene expression and protein levels of two major HS carriers were also determined and revealed that EXTL2 KO promoted significant alterations in HSPG core protein levels. Furthermore, we addressed the effects of aberrant GAGosylation over tumour cells motility and signalling events. Cellular functional analysis demonstrated that EXTL2 KO and concomitant HS increase promoted motile and invasive phenotypes, as well as the activation of key cell surface receptor tyrosine kinase. Overall, our results reveal the crucial roles of EXTL2 and EXTL3 in the modulation of proteoglycans expression and aberrant glycosylation profiles displayed by cancer cells, and the functional impact of these alterations on cells malignant behaviour, further supporting the clinical potential of HS biosynthetic machinery in cancer therapy.