The extracellular matrix mechanics in the vasculature

Abstract

Mechanical stimuli from the extracellular matrix (ECM) modulate vascular differentiation, morphogenesis and dysfunction of the vasculature. With innovation in measurements, we can better characterize vascular microenvironment mechanics in health and disease. Recent advances in material sciences and stem cell biology enable us to accurately recapitulate the complex and dynamic ECM mechanical microenvironment for in vitro studies. These biomimetic approaches help us understand the signaling pathways in disease pathologies, identify therapeutic targets, build tissue replacement and activate tissue regeneration. This Review analyzes how ECM mechanics regulate vascular homeostasis and dysfunction. We highlight approaches to examine ECM mechanics at tissue and cellular levels, focusing on how mechanical interactions between cells and the ECM regulate vascular phenotype, especially under certain pathological conditions. Finally, we explore the development of biomaterials to emulate, measure and alter the physical microenvironment of pathological ECM to understand cell–ECM mechanical interactions toward the development of therapeutics.

By adulthood, the average human will possess over 60,000 miles of blood vessels, encompassing arteries, veins and capillaries. Vascular homeostasis is maintained by resident cell types that regulate ECM composition and structure in response to mechanical and biochemical stimuli that emerge during development and aging. The major vascular cell types are endothelial cells that line the tunica intimate (lumen), vascular smooth muscle cells (vSMCs) that are the most abundant cells in the tunica media (vessel wall) and fibroblasts in the adventitia. Pericytes wrap around endothelial cells in capillaries and post-capillary venules that do not contain vSMCs. Dynamic reciprocity between the cells and the ECM is responsible for vascular homeostasis and disease and also defines arterial stiffness or compliance, a key index of overall cardiovascular health.

Bulk tissue stiffness increases with age and is accelerated by atherosclerosis, hypertension, obesity and diabetes¹. This stiffening has been associated with endothelial dysfunction, fragmented elastin and increased collagen content, and engagement of the immune system. Moreover, the vasculature is exposed to bulk mechanical loading and unloading due to the pulsatile beating of the heart and alterations to the mechanical microenvironment arising from changes in the composition and architecture of the ECM. Overall vascular mechanics and function are the sum total of these cellular and ECM contributions, governed by complex and overlapping pathways. Therefore, both vascular cells and ECM are clinically relevant targets for understanding vascular homeostasis and disease². Thus, a better understanding of how mechanical stimulations impact cell–ECM dynamics will help to clarify vascular pathobiology and develop targeted interventions.

Advances in stem cell and biomaterial research offer promising approaches for modeling the cell–ECM interactions that govern changes in vascular mechanics during health and disease. Biomaterials provide both biocompatible materials for treating disease and adjustable scaffolds for ECM modeling outside of the body³. This Review outlines ECM structure in the vasculature, the cellular interactions that govern bulk vascular mechanics and the use of biomaterials and techniques to study these processes from the organismal to the subcellular level. While this Review focuses on cell–ECM interactions, it must be noted that cell–cell interactions are also crucial for normal vascular function. For example, vasoreactivity relies on signaling between endothelial cells and vSMCs and pericytes, while inflammation is mediated by interactions of endothelial cells, smooth muscle cells (SMCs) and immune cells^4,5. Vascular cells also have intrinsic stiffness, which directly contributes to overall mechanical behavior independent of the ECM.

ECM mechanics in blood vessels

ECM properties, in tandem with blood flow (that is, blood pressure and shear stress) and resting tone, define overall vascular mechanics. An intricate network of structural proteins, the ECM acts as a scaffold to support cells comprising the tissue, provides mechanical integrity and actively modulates homeostasis^6–8. In turn, resident vascular cells modulate the ECM locally, impacting cellular responses via dynamic cell–ECM crosstalk^9–11. By fine-tuning ECM composition, the vasculature of different sizes can effectively serve their physiological roles, including elastic recoil in the large arteries, vascular resistance in arterioles and nutrient transport in capillaries.

Assessing the mechanical behavior of the vasculature and its ECM, both clinically and experimentally, relies on understanding key measures that are governed by Hooke’s law, the first-order linear approximation of elastic bodies to applied forces, stating F = kx, where F is the applied force, k is the stiffness and x represents the deformation. These include but are not limited to stiffness (resistance to elastic deformation when a force is applied: force versus displacement), compliance (ability to dilate in response to increasing hoop stress; inverse of stiffness), elasticity (ability to return to original shape following loss of stress), ultimate strength (maximum stress before failure: force versus area) and viscoelasticity (rate-dependent response to applied stress or strain). In addition, systemic vascular resistance (impedance of blood flow largely driven by arteries and arterioles: pressure versus flow) regulates healthy vascular pressure. Increased stiffness or loss of compliance is a feature of cardiovascular decline and is an independent risk factor for major adverse cardiovascular events. This change in the integrated bulk mechanical properties (arterial stiffening) involves profound changes to the ECM at the microscale^12–15.

Vascular ECM composition

The vascular ECM comprises hundreds of proteins, including collagen, elastin and proteoglycans. The composition and resultant mechanics of the vascular ECM are context specific. Synthesis, deposition, degradation and cross-linkage of these ECM structural proteins are tightly coordinated processes involving a host of enzymes, including matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), a disintegrin and metalloproteinase (ADAM) proteins, transglutaminases and the lysyl oxidase family. While the exact mechanisms are still being studied, resident cells are assumed to be mainly responsible for maintaining their associated ECM.

Endothelial cells are supported by a thin, specialized ECM called the basement membrane (BM) comprising mainly laminin, collagen types IV, XV and XVIII, fibronectin, the glycoprotein nidogen and the heparan sulfate proteoglycan perlecan¹⁶. Collagen IV is cross-linked to assembled laminin via glycoproteins with high affinity for both¹⁷. Collagen provides structural support, while laminin contains binding sites for growth factors. Many investigations into BM mechanics have focused on ocular tissue. Studies using transmission electron microscopy have reported BM thicknesses under 100 nm. However, sample dehydration required for transmission electron microscopy likely introduces artifacts as ocular tissue retains large amounts of water in vivo. Approaches such as atomic force microscopy (AFM), which can be used on hydrated BM, have shown BM thicknesses up to 500 nm¹⁷. The BM elastic modulus is also context dependent, ranging from 0.5 kPa to 5 MPa¹⁷. Densely packed proteins contribute to BM function as a barrier to large molecules and cells. In the aorta, the BM has been reported to be 500 nm, while, in the saphenous vein, the BM is ~100 nm¹⁷. Proper transduction of mechanical stimuli by endothelial cells depends on anchorage to the BM, meaning that the BM can also indirectly impact vascular homeostasis. Collagen IV, laminin and perlecan have also been shown to inhibit vSMC proliferation, support contractile phenotype and minimize low-density lipoprotein uptake^16,18. Thus, BM composition promotes endothelial cell adhesion and tight junction formation and ‘discourages’ pathologic SMC proliferation and foam cell formation. In the microvasculature and developing blood vessels, pericyte recruitment, mediated by endothelial cell-produced platelet-derived growth factor (PDGF), stabilizes the BM^16,19. Furthermore, stimuli indirectly change BM thickness and composition through modulation of endothelial cell and pericyte proteolytic activity²⁰. The vascular BM is crucial for proper endothelial cell function and behavior, including barrier function, permeability, adhesion and migration and paracrine signaling to other cell types¹⁶. Table 1 summarizes differences in types of BMs across species.

Table 1 |.

Overview of the properties of different BMs

Location	Species	Modulus	Dimensions	Componentsa	Notes	Refs.
Retinal BM (epithelial)	Mouse	3–4 MPa	Thickness: 75 nm	Vitronectin		17
Vascular BM	Macaque		Pores: 45–60 nm Fibers: 25–30 nm Thickness: 100–300 nm	Thrombospondin Osteonectin α4/α5 laminin	Reflects non-aorta vessels	17,169
Aortic BM	Macaque		Pores: 55–65 nm Fibers: 30 nm Thickness: 500 nm	Thrombospondin Osteonectin α4/α5 laminin		17,169
Descemet’s (epithelial)	Human	50 kPa	Pores: 60–120 nm Fibers: 30–60 nm Thickness: 130 nm	Collagen VIII	BM between corneal stroma and endothelial layer	17,170
Internal limiting membrane	Human	1.5–5 MPa	Pores: 10–25 nm Fibers: 70–400 nm Thickness: 0.4–10 μm		BM between retina and vitreous body	17
Retinal BM (epithelial)	Human		Thickness: 300 nm	Vitronectin		17
Glomerular BM (endothelial)	Rat	2–5 kPa	Pores: 10–20 nm Fibers: 5–10 nm Thickness: 50–100 nm			17

^aAll BM contains high levels (>50%) of collagen IV as well as perlecan, nidogen, laminin and fibronectin: the highlighted components distinguish the relevant BM from other types.

In the medial layer of large elastic arteries, fibrillar collagens I, III and V and non-fibrillar collagens IV and VI are important. As the medial layer forms the majority of the mass in elastic vessels, understanding the mechanical profile of this layer is a valuable tool in cardiovascular disease (CVD) progression.

Cross-linking of ECM proteins by matrix-remodeling enzymes increases arterial stiffness and ultimate tensile strength. Elastin, an aptly named multimeric protein produced by resident vSMCs, supports elastic deformation and recoil in response to natural fluctuations in blood pressure. The bulk mechanical behavior of arteries can be attributed largely to elastin and collagen content²¹. In humans, Young’s modulus is reported to be 0.3–1.0 MPa for elastin, 100–1,000 MPa for collagen and 1–5 MPa overall for intact arteries²². While comparisons across species and studies are difficult to make, research suggests that aortic circumferential stiffness is higher in young mice than in young humans and that increases with age are observed in both species, although more notably in mice. Additionally, axial stiffness is greater in mice than in humans across age groups²³.

Mechanical cues are important mediators of vascular function in health and disease. In addition to ECM composition and stiffness²⁴, vascular cells respond to transmural pressure, circumferential strain²⁵ and fluid shear stress²⁶. These inputs are transmitted into the cell via the cytoskeleton and associated membrane proteins to modulate vascular function and behavior, including vasoreactivity, proliferation and ECM metabolism (Fig. 1).

Fig. 1 |. Mechanotransduction in vascular cells.

Endothelial cells (ECs), pericytes (PCs) and vSMCs rely on a wide array of signaling molecules, transmembrane proteins and cytoskeletal signal transducers to modulate their phenotypes and ECM regulation. Some proteins are expressed in one or two cell types but not the others, and others are expressed in slightly different forms or abundances (such as integrins or vimentin)^18,179–181. CFTR, cystic fibrosis transmembrane receptor; ICAM, intercellular adhesion molecule; IF, intermediate filament; MT, microtubule; NMDAR, N-methyl-d-aspartate receptor; PECAM, platelet and endothelial cell adhesion molecule; SMA, smooth muscle actin; SMMHC, smooth muscle myosin heavy chain; SUN, Sad1p/UNC-84 domain containing; TRPV, transient receptor potential vanilloid.

Pathologic ECM regulation in vascular disease

Arterial stiffening occurs with aging, independent of other risk factors, and is accelerated by diabetes, hypertension and atherosclerosis. Thus, the relationship between aging and these seemingly distinct CVDs is better understood when viewing pathological ECM remodeling as a unifying theme^2,27.

Pathological remodeling of the microvascular ECM includes collagen IV accumulation and fat deposition resulting in BM thickening, vessel widening and fibrous tissue deposition²⁸. These changes alter integrin-binding site distribution and impair endothelial cell adhesion and tight junction formation, resulting in increased permeability and inflammation²⁹.

In the large arteries, elastin fragmentation and diminished elastin/collagen ratio in the wall are hallmarks of aging and arterial stiffening. While arterioles contain smaller amounts of elastin, vSMCs and their ECM account for their bulk mechanical properties as well. Therefore, vSMCs are an essential target for understanding age-related vascular stiffening. Interactions between endothelial cells and vSMCs are also important when considering arterial stiffening. These interactions can be direct (that is, adhesion molecule N-cadherin) or indirect (that is endothelial cell-derived NO). Examining endothelial cell and vSMC crosstalk thus depends on the type of interaction. For direct interactions, confocal microscopy is well suited for visualizing membrane-bound adhesion molecules, and western blotting can quantify expression levels in tissue or isolated cells. Approaches to assess the indirect interactions include NO assay kits, northern blotting for microRNA and electron microscopy or AFM to examine extracellular vesicles and ECM structure. Whether aberrant cellular behavior precedes or follows pathologic ECM regulation is an important unresolved question, partly due to harmful feedback loops in vascular pathobiology^30,31.

ECM turnover requires synthesizing, depositing and cross-linking new proteins into the existing ECM. Numerous cell- and tissue-level changes occur in the vascular ECM during aging, hypertension, type 2 diabetes and atherosclerosis (Fig. 2) and drive vascular pathologies. ECM remodeling provides a direct link between cellular behavior and changes in mechanical properties. Chronic inflammation in the microvasculature reduces desmin content in the BM, which impairs EC–pericyte adhesion and capillary integrity²⁸. In diabetes, excess glucose accelerates and exacerbates this decline as exemplified in diabetic retinopathy–neuropathy. This, in part, explains the rapid disease progression in older patients with diabetes. Excess glucose also results in advanced glycation end products (AGEs): covalent cross-linkages between sugars and proteins, lipids or nucleic acids²⁷. AGEs cannot be enzymatically cleaved, causing direct stiffening of microvascular ECM²⁷. Additionally, excess glucose reduces pericyte coverage and endothelial cell barrier function, causing leaky, inflamed vessels^19,32. In early-stage atherosclerosis, endothelial barrier disruption causes lipid accumulation in the subendothelium. As the disease progresses, a fibrous cap consisting of fibrillar collagens and fibronectin surrounds the plaque, increasing thickness and stiffness of the vessel wall.

Fig. 2 |. Overview of the overlapping pathologic changes in vascular cells and their ECM.

During vascular aging and disease, changes in cell behavior and local tissue mechanics result in pathological remodeling of the vascular ECM. Dysregulated ECM turnover and increased proliferation of SMCs are common underlying behaviors in pathologic ECM remodeling in the vasculature^5,21,51,182. ECs, endothelial cells; EndMT, endothelial–mesenchymal transition; GAG, glycosaminoglycan; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; SASP, senescence-associated secretory phenotype.

Aging and hypertension are associated with increased expression of MMP-2, membrane type 1 (MT1)-MMP (elastases) and MMP-9 (gelatinase)^33–35 as well as matrix cross-linking enzymes lysyl oxidase and LOXL2 (ref. 36) and TG2³⁷, resulting in marked vessel remodeling. The resultant change in ECM composition and stiffness is mediated by resident cells and feeds back to promote additional cellular derangements. When vSMCs cannot contact intact elastin, they shift toward a synthetic phenotype, increasing cell migration and collagen synthesis, causing a vicious cycle of additional ECM stiffening and cellular dysfunction^27,31. These changes exacerbate chronic inflammation and disruption of endothelial integrity³⁸. Shear stress is also critical in atherosclerosis; endothelial cells exposed to turbulent (as opposed to laminar) flow exhibit increased senescence, disrupted cell–cell junctions and cytoskeletal organization and dysregulation of NOS3 and SOD1 (superoxide dismutase), which are involved in essential anti-inflammatory pathways³⁸. Turbulent flow occurs in atheroprone regions (branch points and curvature) of the vasculature. Thus, a better understanding of cell–cell and cell–ECM crosstalk that drives changes in vascular mechanics is integral to developing CVD and vascular aging models. Below, we highlight pathways that have a role in mechanotransduction, tone and the regulation of vSMC phenotype.

Mechanotransduction in vascular health.

Mechanotransduction is a process in which mechanical stimuli are converted into changes in cell phenotype. While this Review is focused on ECM–integrin signaling, there are different types of mechanoreceptor (Fig. 1) that respond to distinct mechanical stimuli or impact different portions of the mechanotransduction pathway (signal sensing, communication and response). Communication between cytoskeletal proteins and ECM occurs at focal adhesions, dense structures that link extracellular and intracellular proteins through specialized transmembrane receptors³⁹. Focal adhesions comprise ECM proteins bound to integrins and their associated intracellular proteins such as vinculin⁴⁰. Integrins, membrane-bound heterodimeric proteins composed of α and β subunits, help translate information from the surrounding environment to the cell and vice versa^18,40. There are 18 α-subunits and eight β-subunits responsible for the 24 human integrin subtypes. These subtypes can be grouped according to the substrate to which they bind: laminin binding (α₃β₁, α₆β₁₀), collagen binding (α₁β₁, α₂β₁), leukocyte binding and Arg–Gly–Asp (RGD) binding. Leukocyte-binding integrins bind intercellular adhesion molecule (ICAM) found on other cell membranes while RGD-binding integrins (α_vβ₁, α_vβ₃, α_vβ₅) recognize a motif common to ECM proteins like vitronectin and fibronectin⁴¹. The intracellular proteins talin and vinculin link the nascent focal adhesion to the actin cytoskeleton before signal transducers, paxillin, focal adhesion kinase (FAK) and SRC family tyrosine kinases activate Rac. This promotes actin polymerization while inhibiting myosin association in contractile cells such as vSMCs⁴². The downstream effects of focal adhesion signaling include activation of the Rho and mitogen-activated protein kinase pathways, among others^43,44. ECM composition and stiffness directly impact the type and quantity of associated focal adhesions; in this way, focal adhesions serve as highly dynamic mechanotransducers of their surrounding ECM, ultimately directing cell fate decisions⁴².

Mechanosensitive ion channels are membrane proteins with activity governed by the local mechanical milieu. These include Ca²⁺ (L-type, N-type, T-type), K⁺ (shaker, K_v1.1, BK) and Na⁺ (Na_v1.5) channels as well as more recently discovered channels such as the transient receptor vanilloid (TRP) and Piezo families⁴⁵. Generally, mechanical deformations in cytoskeletal or cell membrane organization, caused by shear flow, changes in focal adhesion binding, etc., result in conformational changes in the channels. As a result, ion flux across the cell membrane (or out of intracellular stores) is altered, resulting in downstream changes to cytoskeletal organization, protein expression and overall cell behavior. The structure and function of these well-studied channels are reviewed elsewhere^45–47.

Small GTP-binding proteins (G proteins) are a superfamily of enzymes, comprising five families, involved in numerous biological pathways. Here, we briefly focus on the effects of the Rho subfamily (Rho, Rac and CDC42) on cytoskeletal organization⁴⁸, as this has been implicated in various CVDs, including atherosclerosis and hypertension. In endothelial cells, increased activation of RhoA disrupts the integrity of the endothelial barrier by suppressing junction proteins⁴⁹. In diabetes, receptors for AGE and RhoA form a complex resulting in continued RhoA activation, increasing the intrinsic cell permeability. Through selective interaction with denatured ECM proteins, ROCK can impact cytoskeletal organization and, ultimately, gene expression in all vascular cell types^50,51. ROCK inhibition via statins has provided promising results in vivo and in vitro, including suppressing intimal hyperplasia in a rabbit model of vein graft disease, decreasing vascular leakage in diabetic mice and reducing the incidence of aortic aneurysm and atherosclerosis⁴⁴.

With their luminal surface directly exposed to blood flow, endothelial cells additionally respond to hemodynamic changes to mediate transport and modulate the behavior of vascular and immune cells. To accomplish these unique functions, endothelial cells express a host of markers, including VE-cadherin (VECAD; adherens junction formation), claudins (tight junctions), integrins β₁, β₃ and α₄β₁ (EC–EC, EC–platelet and EC–pericyte adhesion, respectively), platelet endothelial cell adhesion molecule-1 (PECAM-1; leukocyte migration) and ICAM-1 (leukocyte recruitment during inflammation). Changes in the expression of these proteins, especially VECAD and PECAM, diminish the ability of endothelial cells to properly orient and stretch when exposed to fluid shear⁵². Endothelial cells also express mechanosensitive ion channels, with the Piezo1 channel being of particular interest.

Vascular tone and contractility.

Ion channels in the plasma membrane of vSMCs define vascular tone in response to various neurohumoral influences. K⁺ and Ca²⁺ channels are well documented in this regard. Recent studies have expanded the list of channels that regulate vascular tone, and, among these, TRPV4 channels have emerged as an important type of mechanosensing transmembrane proteins^53,54. The larger family of TRP proteins functions as a non-specific Na⁺–Ca²⁺ ion channel in several cells. These are important regulators of vascular tone, given their sensitivity to mechanical stimuli such as cell swelling and shear stress, in addition to chemical stimuli⁵³. TRPV4 channels in vSMCs promote the release of intracellular Ca²⁺ stores, causing smooth muscle hyperpolarization and vasodilation. In endothelial cells, fluid shear stress increases the agonist sensitivity of TRPV4 and closely links TRPV4 to acetylcholine-mediated vasodilation⁵⁵. In addition to conferring Ca²⁺ sensitivity to contractile cells, TRPV4 has been associated with MMP-2 and MMP-9 activation and the suppression of their inhibitor, TIMP-2, suggesting that TRPV4 can promote ECM turnover. However, it has also been shown to regulate transforming growth factor-β (TGFβ) by modulating downstream pathways, including the previously mentioned phosphoinositide 3-kinase (PI3K) and Rho–Ras pathways. In this context, TRPV4 exacerbates excess tissue deposition. Furthermore, mechanical (but not chemical) interactions between integrins and TRPV4 channels serve as a bidirectional regulator of ECM remodeling; TRPV4 channels are rapidly included in focal adhesions, and suppression of β₁-integrins disrupts TRPV4 functioning. Further work elucidating the cell type- and context-dependent nature of TRPV4 signaling may prove invaluable in managing pathologic vascular ECM remodeling.

The Rho family of enzymes is also involved in regulating vascular tone. Rho-associated kinases (ROCKs) in complex with Rho promote vSMC contractility by increasing myosin light chain phosphorylation⁴⁴ without substantial changes in Ca²⁺ concentration. Rho–ROCK promotes contractile phenotype by regulating the actin cytoskeleton and downstream impacts on myocardin-related transcription factors in vSMCs and pericytes⁵⁶. Endothelial cells have a crucial role in regulating vascular tone. They modulate the bioavailability of NO and other small molecules, directing the contraction (or relaxation) of SMCs and pericytes. The expression of angiotensin-converting enzyme (ACE) and ACE2 is more prevalent in endothelial cells of small arteries or arterioles, whereas endothelial nitric oxide synthase (eNOS) is highly expressed in the endothelial cells of large conduit arteries^57,58.

vSMC phenotype regulation.

De-differentiation of vSMCs from a contractile to a synthetic phenotype is associated with activation of several pro-fibrotic pathways and drives a large portion of ECM accumulation in the vasculature. Although the negative impact of TGF-β is well documented⁵⁹, recent work has turned toward pathways with less detrimental effects when inhibited. For example, activation of intracellular PI3K increases vSMC proliferation, migration and ECM synthesis and secretion⁶⁰. The resultant excess collagen deposition predisposes the vascular wall to atherosclerotic lesions. PI3K influences other vascular cell types. Angiogenic retinal pericytes display high PI3K signaling, while inhibition of PI3Kβ causes pericytes to mature and fully embed in their associated BM⁶¹. PI3K signaling in endothelial cells results in increased apoptosis, promotes monocyte adhesion and confers resistance to oxidation⁶⁰. Thus, targeting PI3K could provide benefits by improving overall cellular health in the vasculature. Other examples include the myocardin family members MRTF-A and MRTF-B that have a reciprocal relationship during SMC phenotype switching. The joint myocardin–SRF (serum response factor) pathway directs phenotype modulation via microRNA species that regulate the transcription of various ECM proteins. The senescence-associated secretory phenotype relates to both SMCs and endothelial cells: as vascular cells senesce, they release ECM proteins, such as collagen, that contribute to vascular stiffening⁶².

While the abovementioned pathways are important in mechanotransduction, tone regulation and ECM modulation, it is not an exhaustive list. The Hippo pathway–YAP–TAZ are critical in adequately functioning endothelial cells⁶³. This pathway relies on tyrosine kinases to coordinate sprouting, branching, lumen formation and remodeling required for angiogenesis⁶³. More generally, nuclear membrane proteins (lamin A and lamin C) facilitate mechanotransduction via perturbations in the nuclear envelope and the associated cytoskeleton⁶². The relevance of any pathway will depend on the cell type, disease and vascular bed under consideration.

Analyzing ECM mechanics and cellular interaction

Analyzing ECM mechanics and cellular interaction

Sophisticated biophysical approaches, some of which include biomaterials, now offer opportunities to measure mechanical interactions between cells and the ECM. These include cell–substrate deformation and the development of traction forces. These forces can be measured at the single-cell or tissue level or in static or physiological culture, allowing us to understand cell–matrix interactions that regulate vascular phenotype in development, regeneration and disease.

Classical approaches to measuring vascular mechanics

Classical approaches to measure the mechanical properties of vasculature include in situ (diagnostic) methods and ex vivo techniques that more directly assess passive mechanics. Pulse wave velocity (PWV) remains the gold standard for in situ assessment of vascular stiffness. For noninvasive measurement, flow velocity spectrograms at two locations are obtained using a Doppler probe, and pulse transit time is simultaneously measured using electrocardiograms^64,65. PWV is calculated by dividing the distance between the locations by the pulse transit time. Stiffer blood vessels lead to faster wave propagation and thus higher PWV. This technique can be readily deployed as a clinical tool, as it is now clear that arterial stiffening is a predictor of CVD risk, independent of traditional risk factors. Recent efforts have established reference normal values of PWV, at least in the white population^64–66.

Ex vivo, bulk vessel stiffness and pressure–dimension relationships can be directly measured by tensile testing and pressure myography. For tensile testing, vascular samples are mounted onto pins through the lumen, and the force required to generate a known uniaxial displacement is measured. Knowing tissue dimensions, the measured force and displacement data are converted to engineering stress–strain curves^22,65. Material properties, including incremental elastic modulus and stress–strain at failure, can be determined. Although simple to perform, this uniaxial method does not mimic the hoop stress that arises in vivo. Pressure myography, in which segments are mounted between two small cannulas, provides a more physiological stress–strain relationship. The biaxial tensile test, which more closely resembles the loading experienced by arteries in vivo, has been used to discern the stress intensity and elastic behaviors of the vascular wall and to develop predictive models^67,68. By perfusing the vessel with vasoregulators and flow to develop shear stress, we can assess functional elements of endothelial cells and vSMCs in the context of physiological transmural pressure⁶⁹. Pressure myography can be technically challenging, particularly when maintaining tissue viability.

The methods discussed above yield invaluable information regarding bulk mechanics but do not capture local or cell–ECM interactions.

Biophysical approaches to measuring ECM forces and characterizing mechanical cell–matrix interactions

Atomic force microscopy.

AFM is a powerful tool for studying cell–matrix’s force–distance interactions in vitro or ex vivo^70–75 (Fig. 3a). In conventional AFM, a cantilever with a tip is scanned over the sample, and the resultant deflection is recorded. The AFM tip deflection vector is linearly dependent on the applied force following Hooke’s law. This provides surface topology and force–distance mechanical behaviors, including material stiffness, viscoelasticity and interfacial adhesion energy at nanoscale resolution. The stiffness of isolated tissues, including blood vessels, cells in culture and polymeric hydrogels, can be measured using AFM^9,72,75–80. By functionalizing the cantilever surface, individual cells can be used as tips, allowing for indirect measurement of the adhesion between a single cell and various substrates, a technique referred to as single-cell force spectroscopy^81–84. In colloidal probe AFM, the cantilever is functionalized with proteins of interest and is used to scan a cell monolayer. This approach is useful for measuring heterogeneity in adhesion forces across a monoculture. Other ECM components and integrin-binding proteins can be used to quantify adhesion to specific transmembrane proteins^73,85–87. AFM and its derivatives have been used to characterize the cardiovascular milieu’s stiffness at nanoscale during dilation and constriction^75,88,89, impaired cortical stiffness in atherosclerosis⁹⁰, regulation of vSMC stiffness and adhesion by drugs⁹¹ and mutations in hypoxia-inducible factor⁹² to the local distribution of VECAD receptors on the surface of endothelial cells⁹³. Combining force spectroscopy with optical microscopy techniques such as total internal reflection fluorescence can quantify and visualize cell surface adhesion in tandem^84,94.

Fig. 3 |. Selected approaches to measure cell–ECM mechanical interactions.

a(i–v), AFM measurements of ECM (a(i)), cell indentation (a(ii)), cell (a(iii)), cell in matrix (a(iv)) and tissue (a(v)). a(vi,vii), AFM maps the topography of Epiflex matrix with fibroblasts⁸⁰ (a(vi)) and its stiffness (a(vii))⁸⁰. b, Left, TFM approach in which fluorescent beads are seeded in the surrounding ECM as mechanical sensors. b, Right, TFM example: immunofluorescence images of endothelial colony-forming cells (green) with beads (purple) in dynamic hydrogels and non-dynamic hydrogels (top) and quantification of mean and maximum displacement and speed of the beads in time-lapse (bottom) (n = 15 cells from biological triplicates)¹⁰⁵. GFP, green fluorescent protein. c, Left, optical tweezer approach to trap and manipulate a bead in the surrounding ECM. c, Right, a confocal microscopy image of beads seeded in the ECM around an MCF7 cell (top), and the complex modulus │G*│ describes the material’s microscale rigidity depending on frequency, where ω is the frequency and A and b are the fit parameters, with power law behavior at high frequencies (bottom). Low-frequency (LF) and high-frequency (HF) regimes are indicated¹¹¹. d(i–v), Brillouin microscopy (d(i)) with an example of the virtually imaged phased array (VIPA) Brillouin spectrum, ν represents measured Brillouin shift (d(ii)) and average Brillouin shift of cells treated with cytochalasin D (Cyto D; red) with respect to the control (ctrl; blue) (d(iii)), along with examples of Brillouin shift maps (d(iv)) and brightfield images of live U87 cells (control and treated with cytochalasin D) (d(v))¹¹³.

While AFM is useful for measuring the stiffness of isolated tissues, there are important considerations when investigating the force–distance mechanics of interfacial viscoelasticity and cell–ECM adhesion. Sensitive cell types such as stem cells are unsuitable as probes in single-cell force spectroscopy⁹⁵. Colloidal probe AFM circumvents this problem but requires the cell monolayer to be exposed to open air, greatly increasing the risk of contamination or damage to the culture⁸⁴. Other types of force microscopy have been developed to measure cell–ECM interactions in a three-dimensional (3D) environment.

Traction force microscopy.

Traction is a physical process in which a tangential force is transmitted across an interface between two bodies through dry friction or an intervening fluid film, resulting in motion, stoppage or transmission of power⁹⁶. During this physical process, the traction force is the force used to generate motion between a body and a tangential surface. Traction force microscopy (TFM) can map the mechanical microenvironment of live cells in vitro and measure local strain fields in situ at the fluid–cell interface^74,97–100 (Fig. 3b). In TFM, fluorescent beads are embedded alongside cells in a soft, deformable substrate of known stiffness. By tracking the displacement of beads in proximity to cells, the strength of the interaction between cells and their surroundings is gauged¹⁰¹. TFM can also track the dynamic changes of traction force, matrix deformation and micro-viscoelasticity of ECM near live cells, including real-time sensing of ECM stress changes around cells^102–106. This strategy has been applied to detect stem cell–ECM mechanical interactions that affect cell fate¹⁰⁷. Light-sheet photonic force optical coherence elastography, a TFM adaptation, is a noninvasive method to quantify 3D imaging of ECM viscoelasticity with cellular-scale resolution and dynamically monitor cell-mediated changes to pericellular viscoelasticity¹⁰⁸. Another TFM derivative, traction force optical coherence microscopy, is a newer technology that provides enhanced spatial coverage and temporal sampling¹⁰⁹. To reduce the noise signal, two-layer elastography TFM is modified TFM that allows simultaneous measurement of the Poisson’s ratio of the substratum while also determining cell-generated forces, thus increasing accuracy¹¹⁰. These new technologies can compute time-lapse ECM deformations resulting from cell forces in 3D culture.

Optical tweezers.

Optical tweezers can measure the forces exerted on cells by the ECM as well as the micro-viscoelasticity of the surrounding ECM in vitro while quantitatively altering the cellular mechanical microenvironment. A focused laser is used to manipulate microbeads in the surrounding ECM, allowing for location-specific measurements of changes in ECM viscoelasticity in vitro (Fig. 3c). This technique has been used to show that changes in ECM viscoelasticity can guide epithelial cell patterning and migration^111,112. Certain coatings allow beads to be endocytosed by cells: manipulating engulfed beads is useful for measuring intracellular stiffness^110,113. Unlike AFM, optical tweezers can measure and alter intracellular and extracellular viscoelasticity and dynamic coupling, where cells modulate their internal viscoelasticity to match the surrounding ECM near the cells and also ECM that is not exposed to the outer surface¹¹¹.

In addition to the above methodologies, confocal Brillouin microscopy is a label-free, non-contact method that allows direct readout of the viscoelastic properties of a material (Fig. 3d). Confocal Brillouin microscopy can be adapted to study the viscoelastic properties of biological samples in 3D¹¹³. Table 2 summarizes selected approaches to characterize mechanical cell–matrix interactions, comparing features of different characterization methods.

Table 2 |.

Summary of selected approaches to characterize mechanical cell–matrix interactions

Methods	ECM	Cells	Measurements	Notes	Refs.
AFM	4,5-dimethoxy 2-nitrobenzyl-aminothiol (DMNBAT)-hyaluronic acid (HA)-methacrylate hydrogels	U373-MG human glioblastoma cells	ECM stiffness	AFM tips contact the sample surface, where small deflections provide nanoscale surface topology.	113
Optical tweezers	fibronectin	Epithelial cells	Forces exerted on cells by the ECM	Energy from highly focused lasers is used to manipulate objectives and measure their force–distance responses.	112
TFM	Collagen I	Epicardial cells	Velocity and displacement	Mechanical sensors (that is, fluorescent beads) are seeded in the substrate and are tracked periodically.	104
AFM	Collagen I, III, IV, V and VI	Fibroblast	Matrix topography and stiffness		80
Light-sheet photonic force optical coherence elastography	Polyacrylamide gels	NIH-3T3 fibroblasts	Interacting force and elasticity		108
Optical tweezers and Brillouin microscopy	Polyacrylamide gels	Human glioblastoma cells	Interacting force	Light scattering within elastic material is combined with traditional confocal microscopy.
Traction force optical coherence microscopy	Matrigel	NIH-3T3 fibroblasts	Traction force, velocity and displacement fields		109
TFM	RGD-modified agarose hydrogels	Murine mesenchymal stem cells	Cell traction force and matrix elastic modulus		107
TFM	Collagen I	NIH-3T3 fibroblasts	Mechanical strain		99
Two-layer elastography TFM	Polyacrylamide gels	Physarum polycephalum plasmodia	Cell–substratum deformation and traction force mapping	TFM that also accounts for Poisson’s ratio (deformation perpendicular to load)	110

Finally, in addition to the direct cell- and tissue-level measurement, applying mechanical stimulation using bioreactors and static and cyclic stretching can reveal mechanosensitive mechanisms of vascular network formation^114,115. This approach allows us to assess the effect of externally applied tensile forces on the morphology of vascular networks formed by endothelial cell-embedded 3D polymeric constructs. The measurements show that force intensity correlated with vascular network quality, demonstrating forces’ regulatory role in angiogenesis and vessel structure manipulation¹¹⁶. There are also new tools that allow the investigation of the combined effect of matrix stiffness and hemodynamic forces. For example, using a combination tool of microfluidics and Ca²⁺ imaging techniques, it was found that the composition of the ECM affects Piezo1 mechanosensitivity to hemodynamic forces¹¹⁷.

One of the limitations of the current approaches for cell–matrix force–distance measurements is the limited range in which measurements can be manipulated, mostly at nanoscale and microscale. Given that the distribution of the matrix can be large and non-uniform, it could result in a less representative measurement. Another limitation is that some contact measurements, such as AFM, likely damage the system’s sterility or even damage tissue or cells, thus preventing measurements over time.

Biomaterials to reproduce pathological ECM mechanics

Polymeric hydrogels to reproduce pathological ECM mechanics

With advancements in material sciences and stem cell biology, biomaterials provide opportunities to understand vascular dysfunction in the mechanically altered ECM.

Tissue-engineered vascular grafts (TEVGs) typically recapitulate the bulk mechanical properties of large elastic vessels, placing less emphasis on cell–ECM interactions^118,119. The ideal TEVGs should meet mechanical and biological performance criteria of high tensile properties and blood-compatible features. Collagen, fibrin, proteoglycan, hyaluronan, fibronectin and elastin are all used as ECM proteins for TEVGs, while expanded polytetrafluoroethylene (ePTFE) and polyglycolic acid (PGA) are popular synthetic alternatives¹¹⁹. TEVGs are developed for therapeutics or to generate in vitro models seeded with vascular cells to study disease mechanisms¹¹⁸. Generating vascular beds, which are dense with small-diameter vessels, requires a different approach. The cells are distributed into the solution of polymeric scaffold materials, allowing them to be embedded in the 3D material after gelation. Collagen, fibrin and modified synthetic polymers are often incorporated into hydrogels or printable bio-inks. Growth factors and peptides can be incorporated into the matrix, achieving diffusion and immobilization of molecules at temporal and spatial gradients. The subsequent direction of growth of nascent vessels can be defined by the gradients of angiogenic growth factors that mediate endothelial cell migration¹²⁰. For example, vascular endothelial growth factor (VEGF) gradients within 3D gelatin–collagen gels achieved by programmed diffusion kinetics in a microfluidic device guide the location and morphology of endothelial sprouting to mimic native tissues¹²¹. Hydrogel biomaterials are used to elucidate pathological ECM remodeling in aging⁹, cancer¹²², injury^123,124 and inflammation¹²⁵. By emulating mechanical properties, such as stiffness and viscoelasticity, of the ECM in vitro, we can better understand developmental processes, homeostasis and disease progression. Fig. 4 highlights some approaches to recapitulating vasculature ex vivo and lists common materials used in their fabrication.

Fig. 4 |. Modeling vascular ECM in both large vessels and the microvasculature.

Approaches include conduits (TEVGs), hydrogels, microfluidics and synthetic scaffolds^121,164,183. PLGA, polylactic-co-glycolic acid; PEG, polyethylene glycol; bFGF, basic fibroblast growth factor; PVA, polyvinyl acid; GelMA, gel methacrylate; PDMS, polydimethylsiloxane; PGF, placental growth factor.

Tuning matrix stiffness and viscoelasticity

There is notable interest in developing materials that can faithfully recapitulate the stiffness and viscoelasticity of the ECM to study their impact on cell behaviors such as cell phenotype, proliferation, migration and differentiation^126,127,128. Viscoelastic materials, such as hydrogels, can store and dissipate externally applied energy through reversible deformations or internal frictional interactions¹²⁹. Static mechanical testing, including stress relaxation and creep tests, can be used to determine deformation material experiences over time under a continuous tensile or compressive load. The corresponding force–distance relation and the energy during the material’s deformation can be measured in vitro by AFM-based force spectroscopy and the creep testing module. Dynamic mechanical testing includes frequency-dependent rheology, which allows us to capture hydrogel’s power law rheological characteristics at different frequency scales, and cyclic loading tests for studying cyclic mechanical behaviors due to fatigue. Correspondingly, indentation and particle-based measurements have been adapted to characterize the viscoelastic properties that affect cell–matrix mechanical interactions, such as cell–substratum deformation at the microscale¹³⁰. These methods involve depth-sensing nanoindentation^131–133 and AFM-based force spectroscopy^9,105. These techniques can determine the stiffness and Young’s modulus of the polymeric material through the relationship between the force and the material’s elastic deformation in vitro. Physiological viscoelastic ECMs can be mechanically remodeled by traction forces exerted by cells and exhibit stress relaxation¹³⁴. During this process, deformation of the ECM comes with the exchange of energy. Cells generate more work on viscoelastic substrates relative to purely elastic substrates¹³⁴. In addition to classic contact measuring methods, newer non-contact in vitro methods, such as resonant acoustic rheometry, have also been applied to precisely measure the mechanical properties of soft and viscoelastic biomaterials like hydrogels. Here, a focused ultrasound creates a microscale perturbation on the surface of a material, and the resultant resonance is recorded. The collected frequency spectrum of the resonant surface waves allows for quantification of viscoelasticity at the cell–ECM interface^135,136.

Using hydrogels, it has been shown that matrix stiffness regulates vascular fate. The compliant matrix induces endothelial cell fate, while a stiffer matrix induces SMC fate^137,138 and determines the contractile or synthetic phenotype of vSMCs¹³⁹. In conjunction with VEGF, matrix stiffness regulates the network assembly of blood and lymphatic vessels^140,141. The stiff tumor matrix has been shown to induce angiogenic outgrowth, invasion and neovessel branching¹⁴². Remodeled matrix stiffness in the progression of tumors regulates the process of neovascularization¹⁴³. The in vitro model of endothelial–stromal cell co-culture within hydrogels demonstrates a correlation between stiffening and capillary morphogenesis^144,145.

A recent study showed that a stiff substrate, which imitates arterial stiffness, can trigger endothelial–mesenchymal transition, a process describing the loss of key endothelial cell markers (such as VECAD and PECAM) and acquisition of a mesenchymal, smooth muscle-like phenotype. This transition is an important contributor to atherosclerosis¹⁴⁶. Hydrogel biomaterials have been developed to recapitulate matrix stiffness and elucidate its impact on vascular cell adhesion, proliferation and phenotype¹⁴⁷. Gelatin-based hypoxia-controllable hydrogels are prepared by conjugating phenol-containing ferulic acid to a gelatin backbone, followed by enzymatic cross-linking using a laccase-mediated reaction that consumes oxygen, resulting in hypoxic conditions within the hydrogel¹⁴⁸. Using this oxygen-controllable hydrogel, the mechanism by which hypoxia, co-jointly with matrix viscoelasticity, guides cluster-based vasculogenesis was uncovered^148–150. Hydrogel systems have been used to study the role of viscoelasticity in cell differentiation^151,152 and migration^129,153. Using hydrogels with identical polymer composition but different cross-linking capacities, it was found that viscoelastic dynamic hydrogels promote rapid cell contractility-mediated integrin clustering, leading to activation of FAKs and matrix remodeling, resulting in vascular morphogenesis in vitro and angiogenesis in vivo¹⁰⁵.

Interfacial mechanics

Rebuilding interfacial adhesion is another critical factor for mimicking pathological cell–ECM interfacial mechanics¹⁵⁴. The interfacial mechanics of the ECM and cells can be modeled through the bionic cell–ECM interface. At the molecular level, interfacial adhesion between cells and their ECM environment includes the van der Waals force, hydrogen bonds and electrostatic interactions^155–157. Bioadhesive hydrogels can connect to the cell surface through interfacial bonding, covalent cross-linking and chain entanglement^158,159. The cell–ECM interfacial adhesion energy, from the molecular level to the tissue level, can be measured by AFM in vitro¹⁶⁰. They can be applied for suture-less wound sealing, filling the volume lost in soft tissue defects while promoting early angiogenesis by improving cell migration¹⁵⁹. Furthermore, increasing interfacial adhesion between hydrogel scaffolds and cell culture substrates can enhance the anchorage of endothelial cells, thus preventing dense cellular aggregates during growth¹⁶¹. These studies deepened the understanding of the regulatory mechanism of ECM mechanics to advance tissue engineering and identify potential therapeutic targets.

Responsive hydrogels

To emulate the change in biophysical complexity of the ECM, researchers have developed responsive hydrogel platforms. These hydrogels allow for a change in ECM property ‘on demand’ in response to an external stimulus such as ultraviolet or visible light irradiation. In a recent study, a fibrillar hydrogel was fabricated, which comprised methacrylate collagen I (Col-MA) and methacrylate hyaluronic acid (HA-MA), that stiffens ‘on demand’. By mixing HA-MA with Col-MA and allowing them to polymerize, the hydrogel can then be incubated with a ruthenium photoinitiator and undergo cross-linking using visible light, thus modulating matrix stiffness⁹. Using this platform, it has been discovered that the contractility of endothelial cell in vascular networks increases when the matrix becomes stiffened. This leads to the disassociation of β-catenin from VECAD-mediated adherens junctions and ultimately disrupts the network⁹. Hyaluronic acid hydrogels have also been applied as a chemically and mechanically tunable system to preserve lymphatic endothelial cell phenotypes. Using a high growth factor concentration in combination with low matrix elasticity, this system generated lymphatic cord-like structures¹⁴⁰. Another study achieved in situ repeatedly reversible dynamic tuning of local hydrogel stiffness. Using a dual-cross-linkable alginate hydrogel, increasing or decreasing the concentration of Ca²⁺, the hydrogel becomes stiffer or softer, respectively, impacting mesenchymal stem cell behaviors¹⁶². Table 3 highlights different types of materials with their compositions and features.

Table 3 |.

Summary of in vitro material platforms with their compositions and mechanism of action

Selected in vitro platforms	Compositions of material	Material mechanism of action	Refs.
Natural materials
Collagen	Type I, III, IV and VI collagen	Recapitulate mechanical environments of the ECM, applied to generate in vitro models seeded with vascular cells to study disease mechanisms	171–173
Oxygen-controllable hydrogel	Gelatin-based hypoxia-controllable hydrogels	Emulate hypoxic environments and, co-jointly with matrix viscoelasticity, guide vasculogenesis	148–150
Responsive hydrogels	Col-MA and HA-MA	Hydrogel stiffening ‘on demand’ impacts endothelial cell behaviors, leading to compromised vascular networks.	9
Dynamic hydrogels	The imine and acylhydrazone bonds were cross-linked by aldehyde groups on multi-aldehyde-modified dextran (Dex-CHO) with the original amino groups and modified acylhydrazide groups on gelatin modified with adipic acid dihydrazide (Gtn-ADH), respectively.	Promote rapid endothelial cell contractility-mediated integrin clustering, leading to activation of focal adhesion kinases and matrix remodeling promoting vasculogenesis/angiogenesis	105
Fibrin		Provide permissive environments for endothelial and mesenchymal progenitor cells to form neovascular networks	171
Elastin		Elastin, along with collagen, are the most abundant ECM proteins in the arterial wall.	174,175
Synthetic materials
Nanofiber–hydrogel composite	Free radical copolymerization of acrylic acid, 2-hydroxyethyl methacrylate and acrylic acid N -hydroxysuccinimide ester	Injectable, fill the volume lost in soft tissue defects while promoting early angiogenesis by improving cell migration	158,159,161
Dual-cross-linkable alginate hydrogel	Oxidation and methacrylation of alginates	In situ stiffening of hydrogels decreases human mesenchymal stem cell and human adipose tissue-derived stromal cell spreading and proliferation, and subsequent softening of hydrogels gives way to an increase in cell spreading and proliferation.	162
Polyurethane	Poly(carbonate urethane) and poly(ether urethane)	Support long-term attachment, proliferation and differentiation of vascular cells in 3D bioreactor-based culture conditions	176,177
Electrospun nanofiber	Poly(l-lactide-co-ε-caprolactone) copolymer	Mimic the nanoscale dimension of native ECM; mechanical properties of this structure are comparable to those of the human coronary artery.	178

Conclusions and perspective

As more studies emphasize the importance of modeling ECM mechanics in the vasculature, they inform future studies of pathologies, regeneration and tissue engineering. With advances in material sciences, the components of hydrogels and their microstructures are becoming increasingly dynamic, refined and diversified. As a result, the range of hydrogel stiffness and viscoelasticity has become broader and more dynamic. This trend allows hydrogel systems to rebuild diversified vascular ECM mechanics in more complicated physical, chemical and biological microenvironments. Thus, hydrogels can reconstruct the pathological microenvironment more accurately. Besides rebuilding the microenvironment, advances in hydrogel composition and diversity of mechanical properties make them useful for drug delivery^163,143 and rapid wound healing¹⁶⁴.

Meanwhile, developing new mechanical measuring tools will further our ability to measure and reproduce vascular ECM characteristics in vitro. Rapid progress continues toward understanding how crosstalk between vascular cells and their ECM alters tissue function. Various techniques are used to measure cell–ECM interaction in the context of vascular mechanics, development and disease progression. Bulk measurements of vascular mechanics using PWV, tensile testing and myography complemented by nanoindentation and disruptive and non-distributive imaging approaches at the cell–ECM interface would uncover molecular mechanisms underlying vascular pathologies. Newer techniques of dynamic 3D imaging of cell–ECM interactions over an extended period, such as combining TFM with supervised machine learning and developing new noninvasive measuring methods, should be considered to simulate and predict cell–ECM mechanical interaction behaviors.

In addition to the measuring tools, new materials enhance our capability to reproduce matrix characteristics in physical and pathological conditions in vitro. Hydrogels are being developed with a broader range of mechanical properties and in situ reversibility to reproduce pathological ECM mechanics better. In addition to modulating the material’s mechanical properties, externally applied forces can be used to study vasculature’s mechanical regulation, ranging from millimetric vessels to microvascular networks¹⁶⁵. Recent studies use 3D bioprinting technology to build up cell-laden hydrogel constructs in which a mechanical force can control the growth of blood vessels in the direction of mechanical stimulation with no branches¹²³.

In studies of ECM mechanics in the vasculature, there are still pending questions to be addressed. Even the most advanced contemporary technology cannot perfectly measure and model the vascular ECM because of its complexity. Blood vessels, especially under pathological conditions, reside in an intricate biological, physical and chemical environment. Simply mimicking one or several aspects of that environment using hydrogel materials cannot recapitulate this complexity. In addition, an adult human contains miles of various types of blood vessels, while studies of vascular mechanics are limited to a very small range of length and space. The missing noninvasive measurement capabilities and current approaches’ size and timescale hinder us from better understanding ECM mechanics in the vasculature. More advanced computational simulation, newer materials and reagents, new noninvasive measurement approaches and long-term clinical trials are needed to address this problem.

Hydrogel materials can mechanically regulate vascular formation by influencing attachment, migration, proliferation and differentiation and impacting individual cell morphing into complex multicellular tissues¹⁰⁵. Thus, controlling the mechanics of the hydrogel system to the specific tissue and injury would allow customized interventions on demand toward therapeutics. For example, hydrogels can be injected into the targeted tissue in vivo to be incorporated into the injured microenvironment, where the mechanical features of the hydrogel regulate cellular responses, leading to healing and tissue regeneration. In addition to cell infiltration, proliferation and vascular morphogenesis, blood flow and the resulting shear stress further regulate cellular responses under the hemodynamic context^166,167. One of the prospects for in vivo vascular therapeutics is engineering mechanical stimulations to modulate the rate of cellular processes and stimulate tissue healing and growth¹⁶⁸, thus mechanically accelerating the neovascularization driven by matrix materials. Future work in recapitulating vascular ECM in large and small vessels should focus on a few specific avenues that can potentially push the field forward in multiple ways. For example, loss of intact elastin as we age harms our large blood vessels and likely drives pathologic remodeling in smaller tissues. Studies focusing on elastogenesis, particularly how to promote this process in vSMCs in vivo or in vitro, will open new therapeutic avenues and the development of more biomimetic vascular grafts. Furthermore, research on the connection between ECM composition and stiffness and how they mediate cell response to mechanical stimuli may help explain vascular cell types’ differing, often opposing, responses to the same stimuli. Finally, understanding the role of ECM mechanics in the function and response of immune cells could uncover its impact on a range of vascular pathologies.

In summary, advances in measuring vascular mechanics will increase our understanding of underlying signaling in vascular homeostasis, disease and regeneration and eventually contribute to developing precision vascular therapy. Developing advanced biomaterials with improved pathological relevance is a promising research course to illuminate how diseased tissue impacts blood vessel function and regeneration and identify potential therapeutics to restore vascular function and advance patient care (Box 1).

Box 1. Summary of the limitations of current vascular mechanic studies.

1. Noninvasive mechanical measurements

Aside from PWV, current methods to measure vascular mechanics are conducted on isolated tissue. This presents limitations for diagnostics and understanding mechanical regulation in the context of the functioning tissue.

2. Recapitulating the in vivo environment

Under pathological conditions, tissue contains intricate biological, physical and chemical environments. Biomaterials are designed to mimic several of these environmental aspects, allowing us to study the role of those specific environmental aspects in disease development and progression. Nonetheless, this practical yet reductionist approach cannot completely recapitulate the tissue environment complexity.

3. Size scale using biophysical approaches

Studies of vascular mechanics are limited in the range of length and space in which vascular mechanics can be measured. Given the non-uniform tissue environment, the limited measurement size scale can result in an inaccurate conclusion at the tissue level.

4. Timescale of studies

The timescale of current vascular mechanic studies is weeks to days. However, potential therapeutics may take much longer than that. Long-term clinical trials will be necessary supplements to the current vascular mechanic studies.