Vascular Nephrology

Posttranscriptional mechanisms in vascular homeostasis: non-coding RNAs

It is not the number of genes that defines human complexity. While humans have little over twenty thousand genes, a water flea has thirty-one thousand genes. What does drive complexity is how genes are “wired” and can optimally act together like a complicated electrical circuit with amplifying transistors and effective feedback mechanisms. Posttranscriptional regulation provides such a coordinate control of gene expression in response to environmental changes or cellular injury. Facilitated by an intricate interplay between non-coding RNAs such as microRNAs (miRNAs) or long non-coding RNAs (lncRNAs) and RNA-binding proteins gene expression is regulated to drive the output of functionally related cellular pathways. Over the years we demonstrated pivotal roles for miRNAs in vascular homeostasis, kidney health and disease. Current studies involve:

• The role of sex-specific posttranscriptional pathways in heart failure with preserved ejection fraction (HFpEF) in the context of the Dutch Heart Foundation supported Queen of Hearts consortium and the role of circulating miRNAs for the progression of HFpEF in kidney disease in the Dutch Heart Foundation sponsored CVON consortium RECONNECT.
• Using endothelial cell- and pericyte- lineage tracing mouse models, we aim to identify lncRNAs that are important in maintaining vascular integrity, in the context of chronic kidney disease, and to determine whether the modulation of lncRNA expression levels can influence microvascular stability.
• The role of circular RNAs (circRNAs) in vascular smooth muscle cells (vSMCs), as in healthy arteries, vSMCs serve to maintain vascular tone, with intrinsic and extrinsic factors influencing their control of blood flow and blood pressure in the coronary and peripheral arteries. Upon vascular injury, vSMCs undergo a well-established phenotypic shift from a contractile to a fibroproliferative, migratory state, a physiologic response that aids in repair of the damaged vessel. This fibroproliferative activity of the VSMC can aid in resolution of initial damage , but is also tightly linked with pathophysiological situations such as coronary artery disease (CAD), in-stent restenosis and peripheral arterial disease (PAD).
• Innovative 3D microvessel-on-a chip models
  In a collaboration between our group, the group of professor Thomas Hankemeier (Analytical BioSciences at the LACDR) and organ-on-a-chip company MIMETAS (Leiden) we recently developed a microfluidics-based, 3D ‘microvessel-on-a-chip’ platform that allows the modeling of patient- or disease-specific human pericyte-stabilized microvessels and allows quantitative and parallel testing of microvascular leakage, angiogenesis and inflammation. We aim to apply our platform to serve as a novel diagnostic and prognostic tool as well as a unique technology to develop therapeutic strategies to combat the loss of microvascular integrity in a broad spectrum of systemic diseases including cardiovascular disease, obesity and its complications.

Vascular access dysfunction remains the Achilles’ heel of hemodialysis, leading to substantial morbidity and mortality. The LUMC has an extensive track record in the field of vascular access, which is organized from without our expertise center, in which nephrologists, vascular surgeons, biologists, technicians and engineers are working together. Our research includes preclinical studies on the pathophysiology of vascular access failure, epidemiological studies on the prevalence of vascular access complications, as well as the development of novel therapeutic strategies to improve the durability of vascular access conduits. The latter includes treatment with liposomal drug-formulations to target vascular inflammation, forearm exercise to enhance maturation, tissue engineered vascular grafts and novel medical devices to improve the functionality of arteriovenous access conduits.

We developed a novel technique to make in situ engineered blood vessels for hemodialysis access. These vascular grafts are currently being evaluated in a clinical trial. Recently, we completed a randomized clinical trial on the efficacy of liposomal prednisolone to enhance maturation of radiocephalic fistulas. Furthermore, we developed a new concept for a dialysis needle with the aim to reduce miscannulations. In addition, we studied long-term outcomes of the various configurations of arteriovenous access conduits and the efficacy of interventions to maintain and restore patency and we explored current strategies regarding vascular access management after transplantation. In addition, the PINCH trial is currently being performed in which the efficacy of pre-operative forearm exercise is evaluated to enhance outward remodeling of fistulas.

Supported by a grant from the Health Technology program LUMC-TU Delft, we are currently developing a device that creates the option to make a dynamic AVF, a vascular access conduit that is solely functional during hemodialysis sessions (collaboration with dr.ir. T. Horeman, TU Delft).

Diabetic nephropathy (DN) is a type of chronic kidney disease (CKD) resulting from diabetes. In patient affected by diabetes, factors such hypertension, high glucose and oxidative stress cause a progressive damage of the tubular and glomerular structures of kidneys, and over time a complete loss of kidney function.
Despite DN being one of the major cause of kidney diseases worldwide, the whole pathophysiology of such disease remains largely unknown. We believe that the composition of the gut microbiota, the group of microorganisms living in the human intestine, is one of the factors playing a pivotal role in the onset and progression of DN. In recent years, gut microbiota composition emerged as crucial factor in modulating human health. It is known that diabetic people normally have a different composition of the gut microbiota when compared to healthy subjects and such difference could contribute to some of the pathological features of diabetes, including kidney disease.

In our group, we focus on the relationship between gut microbiota and DN, trying to unravel the relationship between the two with both in vitro and in vivo experiments. The first ones include glomerular endothelial cells and other different cell types, exposed to a wide range of gut microbiota-derived metabolites, in order to better understand the molecular mechanisms underlying the effect of such metabolites. On the other hand, with animal experiments we aim to show the direct influence of different gut microbiota composition on DN. For this reason, we are working on developing a mouse model of DN, followed by transplantation of different types of gut microbiota. In this way, we hope to learn more about the factors that cause kidney disease in diabetic patients.

Chronic kidney disease (CKD), with diabetes mellitus predominating its etiology (diabetic nephropathy), is a leading cause of death due to cardiovascular disease. It is becoming increasingly apparent that post-transcriptional regulation by non-coding RNAs (ncRNAs, including microRNAs and long noncoding RNAs) allow fine-tuning of gene expression and is essential in the pathophysiology of chronic kidney disease (CKD). Despite their intracellular functions, ncRNAs are also present in the bloodstream carried by extracellular vesicles, while miRNAs are also carried by the RNA-binding protein Argonaute2 (Ago2) or lipoprotein complexes such as high-density lipoprotein (HDL).. These systemic, circulating ncRNAs can be horizontally transferred to vascular cells thereby actively affecting their phenotype indicating an active involvement in the pathophysiology of CKD. Our group also showed that CKD leads to a shift in the distribution of circulating ncRNAs among its carriers and exert carrier-dependent functions. In addition, the levels and distribution of ncRNA in particular carriers may provide additional value as biomarker for the development of CKD and its complications.

As such, in close collaboration with the clinic, in a variety of patient cohorts including diabetic nephropathy and older patients reaching end-stage renal disease, we aim to identify causal relationships between circulating ncRNAs and vascular dysfunction and identify (causal) biomarkers for the early detection of CKD and vascular complications.

Platelets represent the second most abundant cell-type in human blood. The primary function of platelets is to survey blood vessels for injury, and to instigate coagulation processes at sites where the endothelium has become damaged. Aside from their haemostatic task, they are also regenerative in nature, as instrumental regulators of angiogenic processes that ultimately serve to repair damaged tissue as a consequence of vascular injury. Importantly, platelets are highly dynamic cells that possess the capacity to respond instantaneously to diverse activating stimuli, triggering their release of a broad range of bioactive molecules including growth factors, chemotactic cytokines and phospholipids serving either hemostatic of regenerative purposes. Our research lines include:

Platelets are instrumental in the mobilization and differentiation of hematopoietic stem cells at the site of a vascular injury.
Activated platelets accumulate at the site of a vascular injury, where they support the escape of the hematopoietic stem cells (HSCs) from flow and provide a pro-angiogenic niche (fertile-soil). Upon immobilization of the HSCs, they become subject to shear stress, which leads to the expression of the receptor for vascular endothelial growth factor receptor (VEGFR), representing a marker of the differentiation/ maturation towards an endothelial cell phenotype. This research focusses on the contribution of platelets in vascular regeneration.

Coagulation, platelets and inflammation in vascular injury and repair
Chronic systemic inflammation and activation of the coagulation system are common mechanistic denominators in microvascular destabilization in kidney transplant patients or patients with atrial fibrillation (AF) or HFpEF. Early papers demonstrated the key role of platelet activation in endothelial progenitor biology and vascular homeostasis., while a seminal paper in the European Heart Journal of revealed the etiology of circulating angiogenic microRNA-126. Also in the context of the Dutch Heart Foundation sponsored CVON consortium RACE V we study the hypothesis that coagulation activation is a causal factor in microvascular rarefaction (loss) in the atria of AF patients thereby leading to the progression of the disease.

The role of Quaking (isoforms) in platelet aggregation
In the process of platelet aggregation, the forming thrombus develops a very distinctive architecture, with a core of densely packed platelets, overlaid by a shell of loosely packed, adherent platelets. The core of the thrombus is consolidated with filamentous actin (F-Actin; red) which gives the thrombus its strength to withstand blood flow. We have studied expression of alternatively spliced isoforms of the RNA-bdining protein Quaking, which may be involved directly or indirectly in F-actin polymerization. These isoforms appeared to be absent in the thrombus core, but highly expressed in the shell. The inventory of expression of proteins involved in F-Actin polymerization and their localization in a thrombus may give insight into the actual processes of vessel sealing. In addition, Quaking is involved in the formation of circRNAs, that are highly enriched in platelets and may represent a novel mechanisms by which Quaking regulates platelet function.