Post-Assembly Modulation of Extracellular Matrix Stiffness in a 3D Human Arteriole Model to Study VSMC Phenotypic Remodeling

1. Overview

Scientific rationale and importance

Arterial stiffening is a defining feature of vascular aging and cardiovascular disease and is closely associated with vascular smooth muscle cell (VSMC) phenotypic remodeling [1]. At the tissue level, increased arterial stiffness arises largely from extracellular matrix (ECM) remodeling, including collagen deposition and crosslinking within the vessel wall. As the ECM stiffens, VSMCs experience a progressively altered mechanical environment that promote phenotypic shifts, including reduced contractile gene expression and activation of synthetic and osteogenic-associated programs [2]. Endothelial cells are also sensitive to changes in matrix mechanics, with increased stiffness influencing junctional organization and barrier stability. Although these stiffness-associated changes are well described in vivo, the direct effects of increased ECM stiffness on VSMCs within an organized human arteriole structure remain difficult to analyze in current in vitro systems.

Recent vascular-on-a-chip models have successfully reproduced perfusion and endothelial (EC) –smooth muscle organization, providing important advances in human vessel modeling [3]. However, in most platforms ECM mechanical properties are fixed at the time of fabrication and cannot be modified once vessels are formed [3]. When stiffness is varied, it is typically achieved by altering collagen concentration or matrix composition prior to assembly. As a result, stiffness cannot be studied as an independent driver of VSMC adaptation within a structured vessel environment.

The goal of this proposal is to develop a human 3D arteriole-on-a-chip in which ECM stiffness can be modulated after formation of a stable EC–VSMC bilayer. By separating vessel assembly from subsequent increases in matrix stiffness, this system will allow direct evaluation of whether increased stiffness alone is sufficient to drive VSMC phenotypic modulation while preserving vascular organization and endothelial integrity. This question is clinically relevant, as arterial stiffening contributes substantially to cardiovascular disease. Defining its direct cellular effects may improve understanding of vascular dysfunction.

Confirmation of funding/facilities/ethics approvals

All work will be conducted using established microfabrication, cell culture, and imaging facilities within the Knight Cancer Precision Biofabrication Hub at Oregon Health & Science University (OHSU). The Biofabrication Hub is equipped for PDMS device fabrication, microfluidics, collagen-based hydrogel systems, and organ-on-chip development, including plasma bonding systems, syringe pumps, biosafety cabinets, CO₂ incubators, and UV/visible light curing systems necessary for post-assembly phototunable collagen crosslinking.

High-resolution imaging and quantitative analysis will be performed using confocal and spinning disk microscopy platforms available through the Advanced Light Microscopy Core at OHSU. Mechanical characterization of hydrogels will be supported by nanoindentation and rheological instrumentation available within the Biofabrication Hub. Statistical consultation is available through the Knight Biostatistics Shared Resource to support experimental design and analysis.

Primary human ECs and VSMCs will be obtained from commercial vendors. No human subjects or animal research is involved; therefore, institutional ethics approval is not required.

Estimated timeline

The proposed work will be completed within 6 months, including device fabrication, vessel formation and stabilization, ECM stiffness modulation, and phenotypic validation.

Data sharing commitment.

All protocols, raw imaging data, and quantified results will be deposited in an open-access repository in accordance with ResearchHub data-sharing requirements.

2. Introduction

VSMCs are central regulators of vessel structure and mechanical stability [4]. In vivo, arterial stiffening develops progressively through ECM remodeling and collagen crosslinking [1, 5, 6]. As matrix stiffness increases, VSMCs experience an altered mechanical environment that promotes phenotypic modulation, including reduced contractile gene expression and activation of remodeling- and calcification-associated programs [2]. These stiffness-associated shifts are key contributors to vascular dysfunction.

Although the relationship between matrix stiffening and VSMC remodeling is well explored in vivo, it remains unclear whether increased ECM stiffness alone is sufficient to initiate these phenotypic changes within an organized human vessel structure. Many studies examine cell responses to substrate stiffness in simplified systems [7, 8], where changes in mechanical properties are often accompanied by alterations in matrix architecture and adhesion signaling. As a result, the direct contribution of stiffness within a structured, multicellular arteriole context remains unresolved.

In vitro vascular microsystems have advanced significantly in recent years and now reproduce perfusion and endothelial–smooth muscle organization under controlled flow [3, 9, 10]. However, progressive vascular stiffening in vivo occurs within an intact vessel architecture [5, 6], and experimental systems that allow controlled modification of ECM stiffness after vessel formation are needed to better model this process. Because endothelial organization and barrier stability are also influenced by matrix mechanics [11], evaluation of VSMC responses within a structured EC–VSMC architecture ensures that stiffness-dependent changes are interpreted within a physiologically relevant context.

Together, this framework enables direct testing of whether increased matrix stiffness alone is sufficient to drive VSMC phenotypic remodeling within a structured human arteriole model.

Background and rationale

Research question

Does post-assembly extracellular matrix stiffening drive phenotypic modulation of vascular smooth muscle cells within an organized human arteriole-on-a-chip while preserving endothelial integrity?

Hypothesis

Increasing perivascular ECM stiffness within a 3D arteriole model will induce VSMC phenotypic modulation, characterized by reduced contractile gene expression and activation of remodeling-associated programs, without disrupting endothelial organization.

Impact statement

This study will establish whether extracellular matrix stiffness can be independently modulated after vessel formation in a 3D human arteriole model and determine the extent to which ECM stiffness alone drives VSMC phenotypic adaptation. By isolating stiffness from changes in matrix composition or vessel geometry, this work provides a more controlled framework for studying vascular mechanobiology. The findings will inform the design of next-generation in vitro vascular systems and support future studies of stiffness-associated vascular remodeling.

3. Methods

Participants/Sample

This study employs a 3D in vitro human arteriole-on-a-chip model. Primary human VSMCs human ECs will be obtained from commercial vendors and used within defined passage ranges (P3–P6). Each experimental condition will include a minimum of n = 5 independent microfluidic devices (biological replicates). Experiments will be repeated across at least two independent cell passages to ensure reproducibility and control for passage-dependent variability. No human subjects or animal models are involved.

Materials and Procedures

Device fabrication vessel formation
Microfluidic arteriole-on-a-chip devices will be fabricated using PDMS (10:1 base:curing agent) cast on custom molds and plasma bonded to glass substrates to form single straight-channel devices. A cylindrical lumen (250 µm diameter) will be generated using acupuncture needle templating. Photo-crosslinkable type I collagen (PhotoCol) will be injected into the device surrounding the templating needle and polymerized at 37°C. Following gel stabilization, the needle will be carefully removed to create a hollow cylindrical lumen within the collagen matrix.

Human VSMCs (mCherry-labeled via lentiviral transduction) will then be seeded onto the inner surface of the collagen lumen to establish a perivascular mural layer, following approaches similar to previously described vascular microsystem protocols [10]. Devices will be cultured for 7 days to allow VSMC attachment, spreading, and matrix remodeling prior to EC seeding.

After VSMC layer stabilization, human ECs (GFP-labeled) will be seeded into the lumen to form a continuous endothelial monolayer. Devices will be maintained under rocker-based bidirectional perfusion to promote endothelial adhesion, circumferential organization, and barrier maturation prior to ECM stiffness modulation. Preliminary studies demonstrate successful formation of a multicellular 3D arteriole architecture with organized VSMC and endothelial layers suitable for post-assembly mechanical modulation (Figure 1)

ECM stiffness modulation
After endothelial stabilization (Day 10–14 of culture), perivascular ECM stiffness will be increased using controlled light-mediated crosslinking of PhotoCol. Devices will be exposed to defined wavelength light (405 nm) at calibrated intensity and duration to achieve reproducible increases in collagen crosslink density while preserving lumen geometry and cellular organization. Collagen concentration, lumen diameter (250 µm), cell source, seeding density, and rocker-based perfusion conditions will be held constant across experimental groups to isolate ECM stiffness as the primary experimental variable.

Matrix stiffness will be validated using mechanical testing of acellular PhotoCol gels prepared under identical crosslinking conditions. Two groups will be evaluated: (1) baseline soft ECM and (2) post-assembly stiffened ECM, targeting an approximately 1.5–3-fold increase in matrix stiffness.

Phenotypic validation

Immunofluorescence staining and confocal microscopy will be used to assess vascular structure and cell phenotype. Endothelial organization will be evaluated using VE-cadherin to assess junctional continuity and monolayer integrity. VSMC phenotype will be assessed using contractile markers (e.g., ACTA2) and markers associated with phenotypic modulation, including the osteogenic-associated transcription factor RUNX2. Marker expression and spatial distribution within the vessel wall will be quantified.

Endothelial barrier function will be evaluated using FITC-dextran (70 kDa) permeability assays. Fluorescent dextran will be perfused through the lumen, and diffusion into the surrounding matrix will be quantified to assess changes in vascular permeability following stiffness modulation.

Planned Analyses

For immunofluorescence experiments, mean fluorescence intensity per device will be quantified and treated as one biological replicate. For FITC-dextran permeability assays, fluorescence intensity in the surrounding collagen matrix will be measured over time, and permeability values will be calculated for each device. Comparisons between soft and stiff ECM conditions will be performed using two-tailed unpaired t-tests following assessment of normality (Shapiro–Wilk test). If data are not normally distributed, Mann–Whitney tests will be used. Statistical significance will be defined as p < 0.05.

Contingent analysis (IF–THEN statements):

IF photo-induced stiffening disrupts vessel structure or cell viability, THEN light intensity and exposure time will be reduced to identify a tolerable stiffness range. IF VSMC phenotypic changes are not observed at the initial stiffness level, THEN the degree of stiffening will be adjusted within a physiologically relevant range. IF endothelial barrier disruption occurs following stiffening, THEN permeability changes will be measured and reported as part of the biological response.

Ethics and Data Management

This study does not involve human subjects or animals and does not require ethical approval. All data will be anonymized at the sample level and deposited in an open-access repository along with protocols and analysis scripts.

4. Pilot Data

Figure 1. Establishment of a 3D human arteriole-on-a-chip with sequential mural and endothelial cell seeding. A) Schematic of device fabrication and vessel formation. Photo cross-linkable type I collagen (PhotoCol, 7 mg/mL) is injected around a templating needle to generate a 250 µm cylindrical lumen. Human VSMCs (mCherry-labeled) are first seeded and cultured for 7 days. Human endothelial cells (GFP-labeled) are then introduced to form a luminal monolayer. After endothelial stabilization, ECM stiffness can be increased via controlled light activation. B) Representative fluorescent images demonstrating cellular organization within the engineered vessel. DAPI (blue) labels nuclei, GFP identifies endothelial cells, and mCherry labels VSMCs. Overlay image demonstrates formation of a multicellular EC–VSMC structure along the collagen lumen. Scale bar = 400 µm.

5. Budget

The proposed budget will support the following categories:

Microfluidic device fabrication materials
Photo-crosslinkable collagen and ECM reagents
Cell culture reagents and media
Primary human ECs and VSMCs
Antibodies and reagents for immunofluorescence
Imaging and data storage costs

Total costs will be adjusted based on early optimization outcomes.

6. References

List all references used in the manuscript using Nature style (e.g. numbered in-text citations and alphabetical references at the end of the manuscript).

1. Mitchell, G.F., Arterial Stiffness in Aging: Does It Have a Place in Clinical Practice?: Recent Advances in Hypertension. Hypertension, 2021. 77(3): p. 768–780.

2. Chen, Y., X. Zhao, and H. Wu, Arterial Stiffness: A Focus on Vascular Calcification and Its Link to Bone Mineralization. Arterioscler Thromb Vasc Biol, 2020. 40(5): p. 1078–1093.

3. Liu, Q., et al., Engineering in vitro vascular microsystems. Microsyst Nanoeng, 2025. 11(1): p. 100.

4. Alford, P.W., et al., Vascular smooth muscle contractility depends on cell shape. Integr Biol (Camb), 2011. 3(11): p. 1063–70.

5. Wagenseil, J.E. and R.P. Mecham, Vascular extracellular matrix and arterial mechanics. Physiol Rev, 2009. 89(3): p. 957–89.

6. Humphrey, J.D., E.R. Dufresne, and M.A. Schwartz, Mechanotransduction and extracellular matrix homeostasis. Nat Rev Mol Cell Biol, 2014. 15(12): p. 802–12.

7. Engler, A.J., et al., Matrix elasticity directs stem cell lineage specification. Cell, 2006. 126(4): p. 677–89.

8. Caliari, S.R. and J.A. Burdick, A practical guide to hydrogels for cell culture. Nat Methods, 2016. 13(5): p. 405–14.

9. Bhatia, S.N. and D.E. Ingber, Microfluidic organs-on-chips. Nat Biotechnol, 2014. 32(8): p. 760–72.

10. Zhu, N., et al., A tissue-engineered endothelial cell - smooth muscle cell arteriole-like model. Biomater Sci, 2025. 13(22): p. 6368–6377.

11. Huynh, J., et al., Age-related intimal stiffening enhances endothelial permeability and leukocyte transmigration. Sci Transl Med, 2011. 3(112): p. 112ra122.

Post-Assembly Modulation of Extracellular Matrix Stiffness in a 3D Human Arteriole Model to Study VSMC Phenotypic Remodeling

Funding Progress

Nonprofit Recipient

Topics

DOI

Post-Assembly Modulation of Extracellular Matrix Stiffness in a 3D Human Arteriole Model to Study VSMC Phenotypic Remodeling

Funding Progress

Nonprofit Recipient

Topics

DOI