Costanza Giampietro

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Giampietro

First Name

Costanza

ORCID

0000-0001-5229-3835

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Publications 1 - 10 of 53

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression
Morini, Marco F.; Giampietro, Costanza; Corada, Monica; et al. (2018)
Circulation Research
Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.
In vitro characterization of endothelial damage from rapid multiaxial mechanical loading
Jakob , Raphael; Narciso , Maria; Choi , Young; et al. (2025)
Acta Biomaterialia
Lining the innermost surfaces of blood vessels, endothelial cells form a monolayer which regulates transport from and to the blood. Under physiological conditions, endothelial cells are exposed to various mechanical stimuli including hydrostatic pressure, wall shear stress, and circumferential stretch due to vessel dilation. In clinical procedures, such as venous graft implantation, mechanical thrombectomy, balloon angioplasty and stenting, acute multiaxial stretching of vessels occurs, leading to endothelial damage. Associated postoperative complications include neointimal hyperplasia and restenosis. In this work, we investigate the formation of damage in endothelial monolayers exposed to a variety of mechanical loading conditions in vitro. To this end, endothelial cells were cultured to form a confluent monolayer on an elastomer substrate, which was then stretched using a new bi-stable device. Endothelial damage, quantified as relative void area, is shown to be much higher in case of equibiaxial stretching than for uniaxial or strip-biaxial stretch states. A discrete network model was developed to rationalize the experimental observations. The computational model allowed to identify the strain energy density as a measure to predict endothelial damage for stretching in arbitrary kinematic states. These results represent an important step towards assessing the risk of iatrogenic endothelial denudation, and providing a criterion for optimization of clinical procedures and devices. Statement of Significance: Endothelial cell integrity is critical for vascular health but is often compromised during interventions such as angioplasty and stenting. These procedures impose acute, multiaxial mechanical loads on the vessel, leading to endothelial denudation — a factor contributing to restenosis. Despite its clinical relevance, the mechanisms leading to such cellular damage remain unclear. In this study, we present an in-vitro platform enabling rapid equibiaxial and strip-biaxial stretching of endothelial monolayers. Our findings demonstrate that equibiaxial loading produces significantly more damage than uniaxial or strip-biaxial loading. By developing a discrete network model, we identify strain energy density as predictive metric for endothelial damage. These insights contribute to a deeper understanding of endothelial mechanobiology and offer a mechanistic framework for optimizing vascular interventions to reduce iatrogenic injury.
Radial matrix constraint influences tissue contraction and promotes maturation of bi-layered skin equivalents
Polak, Jessica; Sachs, David; Scherrer, Nino; et al. (2024)
Biomaterials Advances
Human skin equivalents (HSEs) serve as important tools for mechanistic studies with human skin cells, drug discovery, pre-clinical applications in the field of tissue engineering and for skin transplantation on skin defects. Besides the cellular and extracellular matrix (ECM) components used for HSEs, physical constraints applied on the scaffold during HSEs maturation influence tissue organization, functionality, and homogeneity. In this study, we introduce a 3D-printed culture insert that exposes bi-layered HSEs to a static radial constraint through matrix adhesion. We examine the effect of various diameters of the ring-shaped culture insert on the HSE's characteristics and compare them to state-of-the-art unconstrained and planar constrained HSEs. We show that radial matrix constraint of HSEs regulates tissue contraction, promotes fibroblast and matrix organization that is similar to human skin in vivo and improves keratinocyte differentiation, epidermal stratification, and basement membrane formation depending on the culture insert diameter. Together, these data demonstrate that the degree of HSE's contraction is an important design consideration in skin tissue engineering. Therefore, this study can help to mimic various in vivo skin conditions and to increase the control of relevant tissue properties.
Stretch-induced damage in endothelial monolayers
Choi, Young; Jakob, Raphael; Ehret, Alexander Edmund; et al. (2024)
Biomaterials Advances
Endothelial cells are constantly exposed to mechanical stimuli, of which mechanical stretch has shown various beneficial or deleterious effects depending on whether loads are within physiological or pathological levels, respectively. Vascular properties change with age, and on a cell-scale, senescence elicits changes in endothelial cell mechanical properties that together can impair its response to stretch. Here, high-rate uniaxial stretch experiments were performed to quantify and compare the stretch-induced damage of monolayers consisting of young, senescent, and aged endothelial populations. The aged and senescent phenotypes were more fragile to stretch-induced damage. Prominent damage was detected by immunofluorescence and scanning electron microscopy as intercellular and intracellular void formation. Damage increased proportionally to the applied level of deformation and, for the aged and senescent phenotype, induced significant detachment of cells at lower levels of stretch compared to the young counterpart. Based on the phenotypic difference in cell-substrate adhesion of senescent cells indicating more mature focal adhesions, a discrete network model of endothelial cells being stretched was developed. The model showed that the more affine deformation of senescent cells increased their intracellular energy, thus enhancing the tendency for cellular damage and impending detachment. Next to quantifying for the first-time critical levels of endothelial stretch, the present results indicate that young cells are more resilient to deformation and that the fragility of senescent cells may be associated with their stronger adhesion to the substrate.
Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin
Bauer, Aline; Zambounis, Lito; Kritikos, Ioannis; et al. (2026)
Journal of Experimental Medicine
Afferent lymphatic vessels (LVs) are present in most vascularized tissues and exert important immune and drainage functions, yet human afferent LVs remain poorly studied. Performing single-cell RNA sequencing of lymphatic endothelial cells (LECs) from human skin and subcutaneous adipose tissue, we identified various LEC subsets, including two valve LEC populations located on the upstream and downstream sides of the valve leaflets. The cell adhesion molecule CD24 emerged as a specific marker of upper valve leaflet LECs in human skin and contributed to lymphatic valve development in murine mesentery. Three-dimensional imaging further revealed several unique features of the human dermal lymphatic network, including a high proportion of LYVE-1⁺ pre-collecting vessels containing intraluminal valves, virtually no collectors, and absence of lymphatic muscle cell coverage. Moreover, LECs in blind-ended capillaries and around valves in pre-collectors displayed mixed junctional and morphological phenotypes. These findings reveal key differences between human and murine dermal afferent lymphatics and provide a deeper understanding of human lymphatic-related (patho)physiological processes.
Injectable Senolytic Hydrogel Depot for the Clearance of Senescent Cells
Garau Paganella, Lorenza; Bovone, Giovanni; Cuni, Filippo; et al. (2025)
Biomacromolecules
Small molecules are frontline therapeutics for many diseases; however, they are often limited by their poor solubility. Therefore, hydrophobic small molecules are often encapsulated or prepared as pure drug nanoparticles. Navitoclax, used to eliminate senescent cells, is one such small molecule that faces challenges in translation due to its hydrophobicity and toxic side effects. Further, as senescent cells exhibit context-dependent pathologic or beneficial properties, it is preferable to eliminate senescent cells locally. To formulate navitoclax and enable local treatment, we designed an injectable hydrogel loaded with navitoclax nanoparticles as a senolytic delivery vehicle. Navitoclax nanoparticles (& Oslash; similar to 110 nm) were prepared via solvent-antisolvent nanoprecipitation and formulated in an injectable polymer-nanoparticle (PNP) hydrogel to create a local senolytic depot. Navitoclax-loaded PNP hydrogels selectively cleared senescent cells in vitro in senescent endothelial monolayers. This work demonstrates the value of formulating lipophilic small molecules and the potential of localized drug delivery strategies to improve senolytic therapies.
Mechanical factors influence Î²-catenin localization and barrier properties
Wu, Xi; Cesarovic, Nikola; Falk, Volkmar; et al. (2024)
Integrative Biology
Mechanical forces are of major importance in regulating vascular homeostasis by influencing endothelial cell behavior and functions. Adherens junctions are critical sites for mechanotransduction in endothelial cells. beta-catenin, a component of adherens junctions and the canonical Wnt signaling pathway, plays a role in mechanoactivation. Evidence suggests that beta-catenin is involved in flow sensing and responds to tensional forces, impacting junction dynamics. The mechanoregulation of beta-catenin signaling is context-dependent, influenced by the type and duration of mechanical loads. In endothelial cells, beta-catenin's nuclear translocation and signaling are influenced by shear stress and strain, affecting endothelial permeability. The study investigates how shear stress, strain, and surface topography impact adherens junction dynamics, regulate beta-catenin localization, and influence endothelial barrier properties.Insight box Mechanical loads are potent regulators of endothelial functions through not completely elucidated mechanisms. Surface topography, wall shear stress and cyclic wall deformation contribute overlapping mechanical stimuli to which endothelial monolayer respond to adapt and maintain barrier functions. The use of custom developed flow chamber and bioreactor allows quantifying the response of mature human endothelial to well-defined wall shear stress and gradients of strain. Here, the mechanoregulation of beta-catenin by substrate topography, wall shear stress, and cyclic stretch is analyzed and linked to the monolayer control of endothelial permeability.
Hydrostatic pressure drives sprouting angiogenesis via adherens junction remodelling and YAP signalling
Al-Nuaimi, Dunja Alexandra; Rütsche, Dominic; Abukar, Asra; et al. (2024)
Communications Biology
Endothelial cell physiology is governed by its unique microenvironment at the interface between blood and tissue. A major contributor to the endothelial biophysical environment is blood hydrostatic pressure, which in mechanical terms applies isotropic compressive stress on the cells. While other mechanical factors, such as shear stress and circumferential stretch, have been extensively studied, little is known about the role of hydrostatic pressure in the regulation of endothelial cell behavior. Here we show that hydrostatic pressure triggers partial and transient endothelial-to-mesenchymal transition in endothelial monolayers of different vascular beds. Values mimicking microvascular pressure environments promote proliferative and migratory behavior and impair barrier properties that are characteristic of a mesenchymal transition, resulting in increased sprouting angiogenesis in 3D organotypic model systems ex vivo and in vitro. Mechanistically, this response is linked to differential cadherin expression at the adherens junctions, and to an increased YAP expression, nuclear localization, and transcriptional activity. Inhibition of YAP transcriptional activity prevents pressure-induced sprouting angiogenesis. Together, this work establishes hydrostatic pressure as a key modulator of endothelial homeostasis and as a crucial component of the endothelial mechanical niche.
Anisotropic topographies restore endothelial monolayer integrity and promote the proliferation of senescent endothelial cells
Exarchos, Vasileios; Neuber, Sebastian; Meyborg, Heike; et al. (2022)
Frontiers in Cardiovascular Medicine
Thrombogenicity remains a major issue in cardiovascular implants (CVIs). Complete surficial coverage of CVIs by a monolayer of endothelial cells (ECs) prior to implantation represents a promising strategy but is hampered by the overall logistical complexity and the high number of cells required. Consequently, extensive cell expansion is necessary, which may eventually lead to replicative senescence. Considering that micro-structured surfaces with anisotropic topography may promote endothelialization, we investigated the impact of gratings on the biomechanical properties and the replicative capacity of senescent ECs. After cultivation on gridded surfaces, the cells showed significant improvements in terms of adherens junction integrity, cell elongation, and orientation of the actin filaments, as well as enhanced yes-associated protein nuclear translocation and cell proliferation. Our data therefore suggest that micro-structured surfaces with anisotropic topographies may improve long-term endothelialization of CVIs.
A free-form patterning method enabling endothelialization under dynamic flow
Wu, Xi; Moimas, Silvia; Hopf, Raoul; et al. (2021)
Biomaterials
Endothelialization strategies aim at protecting the surface of cardiovascular devices upon their interaction with blood by the generation and maintenance of a mature monolayer of endothelial cells. Rational engineering of the surface micro-topography at the luminal interface provides a powerful access point to support the survival of a living endothelium under the challenging hemodynamic conditions created by the implant deployment and function. Surface structuring protocols must however be adapted to the complex, non-planar architecture of the target device precluding the use of standard lithographic approaches. Here, a novel patterning method, harnessing the condensation and evaporation of water droplets on a curing liquid elastomer, is developed to introduce arrays of microscale wells on the surface of a biocompatible silicon layer. The resulting topographies support the in vitro generation of mature human endothelia and their maintenance under dynamic changes of flow direction or magnitude, greatly outperforming identical, but flat substrates. The structuring approach is additionally demonstrated on non-planar interfaces yielding comparable topographies. The intrinsically freeform patterning is therefore compatible with a complete and stable endothelialization of complex luminal interfaces in cardiovascular implants.

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Costanza Giampietro

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