Determination of critical shear stress for maturation of human pluripotent stem cell-derived endothelial cells towards an arterial subtype.

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm 2 . We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm 2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm 2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm 2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.

Contribution of actin filaments and microtubules to cell elongation and alignment depends on the grating depth of microgratings.

BACKGROUND: It has been reported that both chemical and physical surface patterns influence cellular behaviors, such as cell alignment and elongation. However, it still remains unclear how actin filament and microtubules (MTs) differentially respond to these patterns. RESULTS: We examined the effects of chemical and physical patterns on cell elongation and alignment by observing actin filament and MTs of retinal pigment epithelium-1(RPE-1) cells, which were cultured on either fibronectin (FN)-line pattern (line width and spacing: 1 µm) or FN-coated 1 µm gratings with two different depths (0.35 or 1 µm). On the surface with either FN-line pattern or micrograting structure, the cell aspect ratios were at least two times higher than those on the surface with no pattern. Cell elongation on the gratings depended on the depth of the gratings. Cell elongation and alignment on both FN-line pattern and 1 µm gratings with 0.35 µm depth were perturbed either by inhibition of actin polymerization or MT depletion, while cell elongation and alignment on 1 µm gratings with 1 µm depth were perturbed only by MT depletion. CONCLUSIONS: Our results suggest that the contribution of actin filaments and MTs to the elongation and alignment of epithelial cells on microgratings depends on the groove depth of these gratings.

The Effects of Biomimetic Surface Topography on Vascular Cells: Implications for Vascular Conduits.

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide and represent a pressing clinical need. Vascular occlusions are the predominant cause of CVD and necessitate surgical interventions such as bypass graft surgery to replace the damaged or obstructed blood vessel with a synthetic conduit. Synthetic small-diameter vascular grafts (sSDVGs) are desired to bypass blood vessels with an inner diameter < 6 millimeters yet have limited use due to unacceptable patency rates. The incorporation of biophysical cues such as topography onto the sSDVG biointerface can be used to mimic the cellular microenvironment and improve outcomes. In this review, the utility of surface topography in sSDVG design is discussed. Firstly, the authors introduce the primary challenges that sSDVGs face and the rationale for utilizing biomimetic topography. The current literature surrounding the effects of topographical cues on vascular cell behavior in vitro is reviewed, providing insight into which features are optimal for application in sSDVGs. The results of studies that have utilized topographically-enhanced sSDVGs in vivo are evaluated. Current challenges and barriers to clinical translation are discussed. Based on the wealth of evidence detailed here, substrate topography offers enormous potential to improve the outcome of sSDVGs and provide therapeutic solutions for CVDs. This article is protected by copyright. All rights reserved.

Testing the Role of Focal Adhesion Kinase (FAK) in Topography-Mediated Stem Cell Differentiation by Inhibiting FAK Phosphorylation.

Stem cell differentiation can be modulated by the substrate topographies. Focal adhesion kinase (FAK) has been identified as a key regulator in topography-induced stem cell mechanotransduction. This chapter will describe a protocol to study the effect of FAK phosphorylation inhibition on topography-mediated stem cell differentiation. The FAK phosphorylation was inhibited using a FAK inhibitor and the effects on stem cell differentiation were examined using western blot and immunofluorescence staining.

Fucoidan and microtopography on polyvinyl alcohol hydrogels guided axons and enhanced neuritogenesis of pheochromocytoma 12 (PC12) cells.

Artificial nerve grafts that support axon growth hold promises in promoting nerve regeneration and function recovery. However, current artificial nerve grafts are insufficient to regenerate axons across long nerve gaps. Specific biochemical and biophysical cues are required to be incorporated to artificial nerve grafts to promote neural cell adhesion and guide neurite outgrowth. Polyvinyl alcohol (PVA) nerve conduits have been clinically approved, but the applicability of PVA nerve conduits is limited to short injuries due to low cell binding. In this study, we explored the incorporation of biochemical cues and topographical cues for promoting neuritogenesis and axon guidance. PVA was conjugated with extracellular matrix proteins and fucoidan, a bioactive sulfated polysaccharide, to improve cell adhesion. Micro-sized topographies, including 1.8 µm convex lenses, 2 µm gratings, and 10 µm gratings were successfully fabricated on PVA by nanofabrication, and the synergistic effects of topography and biochemical molecules on pheochromocytoma 12 (PC12) neuritogenesis and neurite alignment were studied. Conjugated fucoidan promoted the percentage of PC12 with neurite outgrowth from 0% to 2.8% and further increased to 5% by presenting laminin on the surface. Additionally, fucoidan was able to bind nerve growth factor (NGF) on the surface and allow for PC12 to extend neurites in NGF-free media. The incorporation of 2 µm gratings could double the percentage of PC12 with neurite outgrowth and neurite length, and guided the neurites to extend along the grating axis. The work presents a promising strategy to enhance neurite formation and axon guidance, presenting significant value in promoting nerve regeneration.

In-Vitro Study of Indium (III) Sulfate-Containing Medium on the Viability and Adhesion Behaviors of Human Dermal Fibroblast on Engineered Surfaces.

Tissues and organs consist of cells organized in specified patterns that support their function, as exemplified by tissues such as skin, muscle, and cornea. It is, therefore, important to understand how external cues, such as engineered surfaces or chemical contaminants, can influence the organization and morphology of cells. In this work, we studied the impact of indium sulfate on human dermal fibroblast (GM5565) viability, production of reactive oxygen species (ROS), morphology, and alignment behavior on tantalum/silicon oxide parallel line/trench surface structures. The viability of cells was measured using the alamarBlue™ Cell Viability Reagent probe, while the ROS levels in cells were quantified using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate. Cell morphology and orientation on the engineered surfaces were characterized using fluorescence confocal and scanning electron microscopy. When cells were cultured in media containing indium (III) sulfate, the average cell viability decreased by as much as ~32% and the concentration of cellular ROS increased. Cell geometry became more circular and compact in the presence of indium sulfate. Even though actin microfilaments continue to preferentially adhere to tantalum-coated trenches in the presence of indium sulfate, the cells are less able to orient along the line axes of the chips. Interestingly, the indium sulfate-induced changes in cell alignment behavior are pattern dependent-a larger proportion of adherent cells on structures with line/trench widths in the range of 1 µm and 10 µm lose the ability to orient themselves, compared to those grown on structures with line widths smaller than 0.5 µm. Our results show that indium sulfate impacts the response of human fibroblasts to the surface structure to which they adhere and underscores the importance of evaluating cell behaviors on textured surfaces, especially in the presence of potential chemical contaminants.

The Combined Effects of Topography and Stiffness on Neuronal Differentiation and Maturation Using a Hydrogel Platform.

Biophysical parameters such as substrate topography and stiffness have been shown independently to elicit profound effects on neuronal differentiation and maturation from neural progenitor cells (NPCs) yet have not been investigated in combination. Here, the effects of various micrograting and stiffness combinations on neuronal differentiation and maturation were investigated using a polyacrylamide and N-acryloyl-6-aminocaproic acid copolymer (PAA-ACA) hydrogel with tunable stiffness. Whole laminin was conjugated onto the PAA-ACA surface indirectly or directly to facilitate long-term mouse and human NPC-derived neuron attachment. Three micrograting dimensions (2-10 µm) were patterned onto gels with varying stiffness (6.1-110.5 kPa) to evaluate the effects of topography, stiffness, and their interaction. The results demonstrate that the extracellular matrix (ECM)-modified PAA-ACA gels support mouse and human neuronal cell attachment throughout the differentiation and maturation stages (14 and 28 days, respectively). The interaction between topography and stiffness is shown to significantly increase the proportion of ß-tubulin III (TUJ1) positive neurons and microtubule associated protein-2 (MAP2) positive neurite branching and length. Thus, the effects of topography and stiffness cannot be imparted. These results provide a novel platform for neural mechanobiology studies and emphasize the utility of optimizing numerous biophysical cues for improved neuronal yield in vitro.

Fucoidan and topography modification improved in situ endothelialization on acellular synthetic vascular grafts.

Thrombogenesis remains the primary failure of synthetic vascular grafts. Endothelial coverage is crucial to provide an antithrombogenic surface. However, most synthetic materials do not support cell adhesion, and transanastomotic endothelial migration is limited. Here, a surface modification strategy using fucoidan and topography was developed to enable fast in situ endothelialization of polyvinyl alcohol, which is not endothelial cell-adhesive. Among three different immobilization approaches compared, conjugation of aminated-fucoidan promoted endothelial monolayer formation while minimizing thrombogenicity in both in vitro platelet rich plasma testing and ex vivo non-human primate shunt assay. Screening of six topographical patterns showed that 2 µm gratings increased endothelial cell migration without inducing inflammation responses of endothelial cells. Mechanistic studies demonstrated that fucoidan could attract fibronectin, enabling integrin binding and focal adhesion formation and activating focal adhesion kinase (FAK) signaling, and 2 µm gratings further enhanced FAK-mediated cell migration. In a clinically relevant rabbit carotid artery end-to-side anastomosis model, 60% in situ endothelialization was observed throughout the entire lumen of 1.7 mm inner diameter modified grafts, compared to 0% of unmodified graft, and the four-week graft patency also increased. This work presents a promising strategy to stimulate in situ endothelialization on synthetic materials for improving long-term performance.

In vivo evaluation of compliance mismatch on intimal hyperplasia formation in small diameter vascular grafts.

Small diameter synthetic vascular grafts have high failure rate due to the thrombosis and intimal hyperplasia formation. Compliance mismatch between the synthetic graft and native artery has been speculated to be one of the main causes of intimal hyperplasia. However, changing the compliance of synthetic materials without altering material chemistry remains a challenge. Here, we used poly(vinyl alcohol) (PVA) hydrogel as a graft material due to its biocompatibility and tunable mechanical properties to investigate the role of graft compliance in the development of intimal hyperplasia and in vivo patency. Two groups of PVA small diameter grafts with low compliance and high compliance were fabricated by dip casting method and implanted in a rabbit carotid artery end-to-side anastomosis model for 4 weeks. We demonstrated that the grafts with compliance that more closely matched with rabbit carotid artery had lower anastomotic intimal hyperplasia formation and higher graft patency compared to low compliance grafts. Overall, this study suggested that reducing the compliance mismatch between the native artery and vascular grafts is beneficial for reducing intimal hyperplasia formation.

Microarray with micro- and nano-topographies enables identification of the optimal topography for directing the differentiation of primary murine neural progenitor cells.

During development and tissue repair, progenitor cells are guided by both biochemical and biophysical cues of their microenvironment, including topographical signals. The topographical cues have been shown to play an important role in controlling the fate of cells. Systematic investigation of topographical structures with different geometries and sizes under the identical experimental conditions on the same chip will enhance the understanding of the role of shape and size in cell-topography interactions. A simple customizable multi-architecture chip (MARC) array is therefore developed to incorporate, on a single chip, distinct topographies of various architectural complexities, including both isotropic and anisotropic features, in nano- to micrometer dimensions, with different aspect ratios and hierarchical structures. Polydimethylsiloxane (PDMS) replicas of MARC are used to investigate the influence of different geometries and sizes in neural differentiation of primary murine neural progenitor cells (mNPCs). Anisotropic gratings (2 µm gratings, 250 nm gratings) and isotropic 1 µm pillars significantly promote differentiation of mNPCs into neurons, as indicated by expression of ß-III-tubulin (59%, 58%, and 58%, respectively, compared to 30% on the control). In contrast, glial differentiation is enhanced on isotropic 2 µm holes and 1 µm pillars. These results illustrate that anisotropic topographies enhance neuronal differentiation while isotropic topographies enhance glial differentiation on the same chip under the same conditions. MARC enables simultaneous cost-effective investigation of multiple topographies, allowing efficient optimization of topographical and biochemical cues to modulate cell differentiation.

Detachment of bovine corneal endothelial cell sheets by cooling-induced surface hydration of poly[(R)-3-hydroxybutyrate]-based thermoresponsive copolymer coating.

Cell sheet technology (CST) is a fascinating scaffoldless tissue engineering technique to generate a physiologically representative tissue replacement from autologous sources. As compared to conventional enzymatic cell harvesting methods, CST enables the preservation of important cell-to-cell junctions and extracellular matrix (ECM) components. However, covalent grafting methods are often employed for CST. In this study, a series of triblock copolymers with a hydrophobic and biocompatible poly[(R)-3-hydroxybutyrate] (PHB) central block flanked by varying lengths of terminal poly(N-isopropylacrylamide) (PNIPAAm) blocks (PNIPAAm-PHB-PNIPAAm) was synthesized via atom transfer radical polymerization of NIPAAm. The thermoresponsive triblock copolymers were explored as a non-covalent surface coating for culturing and detaching bovine corneal endothelial cell (BCEC) sheets. Aqueous solutions of the triblock copolymers produced thermosensitive micelles which can be drop-casted on glass substrates, resulting in a temperature-responsive surface. Importantly, incorporating a central hydrophobic PHB block enabled the anchoring of the coating to the bare substrate and enhanced the proliferation rate of the BCECs studied. Effective detachment of an intact cell sheet was also demonstrated via a cooling treatment at 4 °C for 20 min, and the viability of the detached cell sheet was found to be unaffected by the cooling. This work may potentially inspire more studies involving the non-covalent thermoresponsive polymer coatings for corneal tissue engineering applications.

Rabbit Surgery Protocol for End-to-End and End-to-Side Vascular Graft Anastomosis.

Preclinical testing in animal model is a required stage of vascular device development. Among small animal models, rabbits provide vasculature with relative larger caliber for anastomotic implantation of vascular grafts as preclinical testing before conducting large animal studies. Rabbits have similar hemostatic mechanism with human and can accommodate vascular grafts with various diameters at different locations, and thus provide a valid model to assess small-diameter vascular grafts. This chapter will describe the procedures and materials required to conduct survival surgery in rabbit carotid artery models for implantation of small-diameter tubular grafts with an end-to-side and end-to-end anastomotic technique.

Hemocompatibility of micropatterned biomaterial surfaces is dependent on topographical feature size.

Small-diameter synthetic vascular grafts that have improved hemocompatibility and patency remain an unmet clinical need due to thrombosis. A surface modification that has potential to attenuate these failure mechanisms while promoting an endothelial layer is the micropatterning of luminal surfaces. Anisotropic features have been shown to downregulate smooth muscle cell proliferation, direct endothelial migration, and attenuate platelet adhesion and activation. However, the effect of micropatterning feature size and orientation relative to whole blood flow has yet to be investigated within a systematic study. In this work, hemocompatibility of micropattern grating sizes of 2, 5, and 10 µm were investigated. The thrombogenicity of the micropattern surface modifications were characterized by quantifying FXIIa activity, fibrin formation, and static platelet adhesion in vitro. Additionally, dynamic platelet attachment and end-point fibrin formation were quantified using an established, flowing whole blood ex vivo non-human primate shunt model without antiplatelet or anticoagulant therapies. We observed a higher trend in platelet attachment and significantly increased fibrin formation for larger features. We then investigated the orientation of 2 µm gratings relative to whole blood flow and found no significant differences between the various orientations for platelet attachment, rate of linear platelet attachment, or end-point fibrin formation. MicroCT analysis of micropatterned grafts was utilized to quantify luminal patency. This work is a significant step in the development of novel synthetic biomaterials with improved understanding of hemocompatibility for use in cardiovascular applications.

Vascular Imaging in Small Animals Using Clinical Ultrasound Scanners.

Conventional ultrasound with frequency (2-15 MHz) has been a global diagnostic and therapeutic tool in clinical medicine, and high-frequency ultrasound (>30 MHz) has been a powerful investigative device for preclinical studies such as cardiovascular research. In this chapter, we describe the use of conventional ultrasound with a 2.5-10 MHz transducer as an investigative device for the measurement/detection of blood flow in rabbit model. The chapter will describe the procedures for the preparation of sonographer, imaging locations, and the details of the rabbits used as well as detailed imaging steps for the preoperative, immediately after operation, and postoperative follow-up ultrasound for vascular surgery, using a vascular graft implantation as an example. We also provide useful notes to avoid pitfalls for successful imaging. The overall goal of this chapter is to deliver the steps in using low-cost, non-invasive, and highly versatile clinical ultrasound imaging in preclinical small animal testing.

Fucoidan for cardiovascular application and the factors mediating its activities.

Fucoidan is a sulfated polysaccharide with various bioactivities. The application of fucoidan in cancer treatment, wound healing, and food industry has been extensively studied. However, the therapeutic value of fucoidan in cardiovascular diseases has been less explored. Increasing number of investigations in the past years have demonstrated the effects of fucoidan on cardiovascular system. In this review, we will focus on the bioactivities related to cardiovascular applications, for example, the modulation functions of fucoidan on coagulation system, inflammation, and vascular cells. Factors mediating those activities will be discussed in detail. Current therapeutic strategies and future opportunities and challenges will be provided to inspire and guide further research.

The effects of surface topography modification on hydrogel properties.

Hydrogel has been an attractive biomaterial for tissue engineering, drug delivery, wound healing, and contact lens materials, due to its outstanding properties, including high water content, transparency, biocompatibility, tissue mechanical matching, and low toxicity. As hydrogel commonly possesses high surface hydrophilicity, chemical modifications have been applied to achieve the optimal surface properties to improve the performance of hydrogels for specific applications. Ideally, the effects of surface modifications would be stable, and the modification would not affect the inherent hydrogel properties. In recent years, a new type of surface modification has been discovered to be able to alter hydrogel properties by physically patterning the hydrogel surfaces with topographies. Such physical patterning methods can also affect hydrogel surface chemical properties, such as protein adsorption, microbial adhesion, and cell response. This review will first summarize the works on developing hydrogel surface patterning methods. The influence of surface topography on interfacial energy and the subsequent effects on protein adsorption, microbial, and cell interactions with patterned hydrogel, with specific examples in biomedical applications, will be discussed. Finally, current problems and future challenges on topographical modification of hydrogels will also be discussed.

POPX2 phosphatase enhances topographical contact guidance for cell morphology and migration.

Topography mediated contact guidance affects multiple cell behaviors such as establishment of cellular morphology and migration. The direction of cell migration is associated with the establishment of cell polarity, which also affects the primary cilia in migrating cells. POPX2, a partner of PIX2, is involved in pathways essential to primary cilium formation, while over-expression of POPX2 has been reported to cause a loss of cell polarity during migration. This study aims to examine how topographical cues direct morphological changes, and how topography affects the process of cellular migration and primary cilium architecture, in the context of POPX2 over-expression. Thus, the effect of anisotropic topography, 2 µm grating pattern on tissue-culture polystyrene, was used as a contact guidance cue to investigate the migration and cell polarity of POPX2 overexpressing cells, in comparison to control NIH3T3 fibroblast cells. We report that POPX2 overexpressing NIH3T3 cells were more sensitive to surface topographical cues as the cells became more elongated. In addition, these cues also affected focal adhesion alignment of POPX2 overexpressing cells. Cell migration was further studied using wound closure assays, in which the 2 µm gratings were designed to be either perpendicular or parallel to wound-induced cell migration direction, which would be agonistic or antagonistic to cell migration, respectively. We observed that both POPX2 overexpressing cells' migration direction and migration rate were more significantly influenced by gratings direction compared to control NIH3T3 cells. The migration paths of POPX2 overexpressing cells become more direct in the presence of anisotropic topographical cues. Further, cilia and centrosome alignment, which is important in cell migration, was also affected by the direction of gratings during this migration process. Collectively, enhancement of NIH3T3 cell sensitivity towards surface topography through POPX2 overexpression might reflect one of the mechanisms that combine biochemical and mechanical cues for directional cell migration.

Enhanced efficiency of nonviral direct neuronal reprogramming on topographical patterns.

Nonviral direct neuronal reprogramming holds significant potential in the fields of tissue engineering and regenerative medicine. However, the issue of low reprogramming efficiency poses a major barrier to its application. We propose that topographical cues, which have been applied successfully to enhance lineage-directed differentiation and multipotent stem cell transdifferentiation, could improve nonviral direct neuronal reprogramming efficiency. To investigate, we used a polymer-BAM (Brn2, Ascl1, Myt1l) factor transfection polypex to reprogram primary mouse embryonic fibroblasts. Using a multiarchitecture chip, we screened for patterns that may improve transfection and/or subsequent induced neuron reprogramming efficiency. Selected patterns were then investigated further by analyzing ß-tubulin III (TUJ1) and microtubule-associated protein 2 (MAP2) protein expression, cell morphology and electrophysiological function of induced neurons. Certain hierarchical topographies, with nanopatterns imprinted on micropatterns, significantly improved the percentage of TUJ1+ and MAP2+ cells. It is postulated that the microscale base pattern enhances initial BAM expression while the nanoscale sub-pattern promotes subsequent maturation. This is because the base pattern alone increased expression of TUJ1 and MAP2, while the nanoscale pattern was the only pattern yielding induced neurons capable of firing multiple action potentials. Nanoscale patterns also produced the highest fraction of cells showing spontaneous synaptic activity. Overall, reprogramming efficiency with one dose of polyplex on hierarchical patterns was comparable to that of five doses without topography. Thus, topography can enhance nonviral direct reprogramming of fibroblasts into induced neurons.

Characterization and Functional Assessment of Endothelial Progenitor Cells in Ischemic Stroke Patients.

Endothelial dysfunction has been implicated in atherosclerosis, ischemic heart disease, and stroke. Endothelial progenitor cells (EPCs), found in the bone marrow and peripheral blood as rare cell population, demonstrated a high proliferation and differentiation capacity. Understanding how such diseases influence the quantity and functionality of EPCs is essential for the development of novel therapies. This study aims to investigate the factors that affect the quantity and functionality of circulating EPCs in stroke patients and healthy controls. Blood samples were collected once from healthy donors (n = 30) and up to 3 times (within 7 days (baseline), 3 and 12 months post-stroke) from stroke patients (n = 207). EPC subpopulations were isolated with flow cytometry for characterization. The Matrigel tubular formation assay was performed as a measure of functionality. An increased amount of circulating EPCs was observed in stroke patients over 45 years when compared to age-matched healthy individuals. EPCs showed a rising trend in stroke patients over the 12-month post-stroke period, reaching statistical significance at 12 months post-stroke. Isolated CD34+KDR+ cells from stroke patients showed impairment in tubular formation capability when compared to cells from healthy donors. The quantity and vasculogenic function of circulating EPCs in peripheral blood have been effectively evaluated in stroke patients and healthy control donors in this study. Age and stroke are found to be 2 influencing factors on the angiogenic capacity. It is suggested that the increase in EPC number is triggered by the recovery response following ischemic stroke. Graphical abstract.

Current understanding of intimal hyperplasia and effect of compliance in synthetic small diameter vascular grafts.

Despite much effort, synthetic small diameter vascular grafts still face limited success due to vascular wall thickening known as intimal hyperplasia (IH). Compliance mismatch between graft and native vessels has been proposed to be one of a key mechanical factors of synthetic vascular grafts that could contribute to the formation of IH. While many methods have been developed to determine compliance both in vivo and in vitro, the effects of compliance mismatch still remain uncertain. This review aims to explain the biomechanical factors that are responsible for the formation and development of IH and their relationship with compliance mismatch. Furthermore, this review will address the current methods used to measure compliance both in vitro and in vivo. Lastly, current limitations in understanding the connection between the compliance of vascular grafts and the role it plays in the development and progression of IH will be discussed.