Physiological Response of Tissue-Engineered Vascular Grafts to Vasoactive Agents in an Ovine Model

Abstract

Tissue-engineered vascular grafts (TEVGs) are emerging as promising alternatives to synthetic grafts, particularly in pediatric cardiovascular surgery. While TEVGs have demonstrated growth potential, compliance, and resistance to calcification, their functional integration into the circulation, especially their ability to respond to physiological stimuli, remains underexplored. Vasoreactivity, the dynamic contraction or dilation of blood vessels in response to vasoactive agents, is a key property of native vessels that affects systemic hemodynamics and long-term vascular function. This study aimed to develop and validate an in vivo protocol to assess the vasoreactive capacity of TEVGs implanted as inferior vena cava (IVC) interposition grafts in a large animal model. Bone marrow–seeded TEVGs were implanted in the thoracic IVC of Dorset sheep. A combination of intravascular ultrasound (IVUS) imaging and invasive hemodynamic monitoring was used to evaluate vessel response to norepinephrine (NE) and sodium nitroprusside (SNP). Cross-sectional luminal area changes were measured using a custom Python-based software package (VIVUS) that leverages deep learning for IVUS image segmentation. Physiological parameters including blood pressure, heart rate, and cardiac output were continuously recorded. NE injections induced significant, dose-dependent vasoconstriction of TEVGs, with peak reductions in luminal area averaging ∼15% and corresponding increases in heart rate and mean arterial pressure. Conversely, SNP did not elicit measurable vasodilation in TEVGs, likely due to structural differences in venous tissue, the low-pressure environment of the thoracic IVC, and systemic confounders. Overall, the TEVGs demonstrated active, rapid, and reversible vasoconstrictive behavior in response to pharmacologic stimuli. This study presents a novel in vivo method for assessing TEVG vasoreactivity using real-time imaging and hemodynamic data. TEVGs possess functional vasoactivity, suggesting they may play an active role in modulating venous return and systemic hemodynamics. These findings are particularly relevant for Fontan patients and other scenarios where dynamic venous regulation is critical. Future work will compare TEVG vasoreactivity with native veins and synthetic grafts to further characterize their physiological integration and potential clinical benefits.

Plain language summary

Impact Statement

This study presents a novel in vivo method to assess the vasoreactivity of tissue-engineered vascular grafts (TEVGs) in a large animal model. By demonstrating that TEVGs can actively constrict in response to pharmacologic stimuli, this work provides new evidence of their functional integration with the circulatory system. These findings advance the field by highlighting the dynamic physiological potential of TEVGs, with important implications for improving long-term outcomes in congenital heart disease, especially in Fontan circulation where controlled venous flow is critical.

Introduction

Vascular grafts are widely used in cardiovascular surgery enabling the replacement, or bypass of diseased, damaged, or congenitally absent blood vessels. Synthetic polymer-based materials, such as expanded polytetrafluoroethylene (ePTFE, GoreTex) and polyethylene terephthalate (PET, Dacron), are traditionally utilized for this purpose. However, despite their widespread clinical application, use of these synthetic materials is not without complication. Calcification, stenosis, compliance mismatch, intimal hyperplasia, and the resulting adverse effects to flow dynamics can ultimately lead to graft failure and the need for reintervention.^1–7 Such complications arise primarily due to differences in the mechanical and functional properties between synthetic grafts and native vessels, creating a mismatch that negatively affects hemodynamics and shear stress at the graft site.

Tissue-engineered vascular grafts (TEVGs) offer an alternative to synthetic vascular grafts. TEVGs are comprised of a polyglycolic acid (PGA) internal mesh scaffold coated on both external and luminal surfaces with a 50:50 copolymer of polycaprolactone (PCL) and polylactic acid (PLA), then seeded with bone marrow-derived mononuclear cells on the day of surgery. They have been implanted into Fontan circulation as extracardiac conduits and inferior vena cava (IVC) interposition grafts.^8–10 Designed to biodegrade completely within 6 months after implantation and be replaced by autologous tissue to create a living vascular conduit, TEVGs offer several advantages over synthetic grafts.^9,11 Neovessels arising from this process of growth and remodeling resemble native vein histologically and have been shown to have growth capacity,¹¹ resist calcification, and retain physiological compliance.⁶ Furthermore, both in vivo and ex vivo testing has shown that TEVGs can mitigate compliance mismatch and improve flow dynamics, leading to better long-term outcomes.^6,9,11,12 Other studies preceding this current work have indicated that TEVGs may harbor vasoreactive functionality comparable to native venous tissue.

Vasoreactivity, the intrinsic capacity of blood vessels to adjust blood flow in response to the diverse physiological demands of the body, is another important characteristic of native vascular tissue. Under physiological conditions, vascular homeostasis is maintained by the activity of endothelial cells induced by various mechanical and chemical stimuli, producing vasoactive molecules that subsequently act on vascular smooth muscle cells.¹³ Vasoreactivity occurring in one vascular distribution may have systemic implications on circulation. For example, arterial vasoconstriction and the associated increase in downstream resistance can lead to elevated cardiac and central venous pressures, as well as increased workload on the heart.¹⁴ On the other hand, venoconstriction can increase mobilization of peripheral blood volume, enhance central venous return, and increase cardiac output in preload-dependent conditions.¹⁵ Meanwhile, vasodilation has been associated with improved blood flow and oxygen delivery, but an excess of this phenomenon, as seen in septic shock, results in hypotension that is detrimental to end-organ perfusion.¹⁶ This interplay between constriction and dilation is therefore carefully regulated by the body’s innate mechanisms.

Historically, vasoreactivity testing has been limited to ex vivo experiments that typically involve the examination of isolated vessel segments treated with pharmacological agents in controlled environments, such as tissue baths.¹¹ Such studies are limited in their translational relevance, particularly when investigating engineered grafts intended for implantation in living systems. While ex vivo testing may allow for controlled analyses of specific pharmacological responses, it does not fully capture TEVG performance under the dynamic conditions of the body, such as pulsatile pressure, neurohumoral regulation, and systemic feedback mechanisms that influence real-time vascular tone.

To date, in vivo vasoreactivity assessments have been primarily conducted in native vessels, such peripheral veins, pulmonary arteries, and coronary arteries, with the goal of evaluating treatment response to various pharmacological agents or establishing a diagnosis.^17–21 Most literature on vasoreactivity testing have focused on hemodynamic changes occurring in vascular beds or the systemic circulation as a whole. Few studies have investigated the vasoactive behavior of a single vessel or implanted graft, leaving a critical gap in our understanding of how vascular conduits such as TEVGs function in vivo over time.^22–25 Therefore, to better evaluate graft functionality in a physiologically relevant setting, we sought to develop a protocol for testing the physiological response of TEVGs implanted as IVC interposition grafts in sheep. By assessing the vasoreactive properties of TEVGs in an in vivo model, we aim to provide deeper insights into their behavior within the systemic circulation and improve our understanding of their long-term performance. Ultimately, this approach may support the design of next-generation vascular grafts that are not only structurally durable but also functionally responsive within the living host.

Materials and Methods

The study protocol was approved by the Institutional Animal Care and Use Committee of Nationwide Children’s Hospital Abigail Wexner Research Institute (AR22-0004). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. Dorset sheep (Ovies aries) with a weight range of 72–120 kg and age range of 4–8 years were housed in a specific pathogen-free environment with free access to food and water for at least one week before the procedure. Animals underwent preoperative assessment by veterinary staff to ensure that they could safely undergo anesthesia. Bone marrow-seeded TEVG scaffolds were implanted in the intrathoracic IVC of sheep between the ages of 4–8 months, as previously described.²⁶

Experiments

Animal preparation

Sheep were fasted for 12 h prior to the administration of anesthesia. They were sedated with a combination of ketamine (4 mg/kg) and diazepam (0.5 mg/kg), injected through the internal jugular vein. Animals were maintained under anesthesia using a combination of inhaled isoflurane 1–3% with 100% oxygen and continuous rate infusion of propofol (20–45 mg/kg/h). A single-lumen venous catheter was placed into the lateral saphenous vein for continuous fluid and propofol administration. Sheep were intubated using a single-lumen endotracheal tube, and an orogastric tube was inserted for decompression of the stomach and rumen. An arterial line was placed in the auricular artery for continuous blood pressure monitoring, and a pulse oximeter was secured to the tongue to monitor oxygen saturation and pulse rate. Electrocardiogram (EKG) leads were placed on all four limbs. In the operating room, the sheep was initially positioned dorsal for the insertion of bilateral catheter sheaths. Two sheaths (7- and 9-Fr) were placed in each internal jugular vein (Fig. 1A, B). A dose of heparin (150 U/kg) was given after placement of all sheaths. A Swan-Ganz catheter was inserted through the left jugular vein to the main pulmonary artery. A 7-Fr multitrack catheter was also inserted through the left jugular vein and into the abdominal inferior vena cava (IVC) just below the IVC hiatus of the diaphragm for drug injection. The sheep was then turned into a left lateral decubitus position for the remainder of the procedure. An intravascular ultrasound (IVUS) catheter was placed through the right internal jugular vein to the midway point of the thoracic IVC interposition graft or an equivalent position in the thoracic IVC of sheep. A second 7-Fr multitrack catheter was positioned similarly for intravenous pressure measurement. Of note, rates of anesthetic administration and ventilator settings were kept constant throughout the procedure to minimize confounding variables.

FIG. 1.

Catheterization procedure for in vivo vasoreactivity. (A) Anteroposterior fluoroscopic view showing catheter placement in the ovine model. A 7 Fr. Multi-Track catheter is introduced for inferior vena cava (IVC) pressure measurements, while another 7 Fr Multi-Track catheter is used for drug injection into the retrohepatic abdominal IVC. A Swan-Ganz catheter is placed for hemodynamic monitoring, and intravascular ultrasound (IVUS) is utilized for continuous vessel imaging. (B). Lateral fluoroscopic view of the catheter placements. (C). Experimental protocol for vasoreactivity testing. Bolus injections of norepinephrine (0.25 and 0.75 mcg/kg) and nitroprusside (10 mcg/kg) are delivered through the injection catheter. Baseline hemodynamic parameters (blood pressure, heart rate, IVC pressure, and cardiac output) are recorded before drug administration, followed by serial and continuous measurements at specific time points postinjection. IVUS recording is performed throughout the experiment. (D). Representative hemodynamic responses to vasoactive drug injections. Cross-sectional area (black), mean arterial pressure (blue), and heart rate (red) are plotted over time.

Vasoconstrictor protocol

The norepinephrine used in this protocol was diluted to a concentration of 10 mcg/mL. Baseline measurements of vital signs including heart rate and blood pressure, midgraft/midthoracic IVC pressure, and cardiac output were recorded prior to drug administration. Three measurements of baseline cardiac output were obtained using the thermodilution method. Continuous cross-sectional imaging of the midgraft/midthoracic IVC at baseline, during drug injection, and after drug injection was obtained using IVUS. First, a low dose of norepinephrine (0.25 mcg/kg) was given as a rapid injection through the left-sided multitrack into the abdominal IVC near the hiatus, followed by a 1.5-mL saline flush. The previously mentioned hemodynamic parameters were recorded 15 s after administration of norepinephrine (the time of maximum drug effect as seen on IVUS), and then every minute for 5 min. The animal was allowed to recover from the drug injection and return their baseline vitals. A high dose of norepinephrine (0.75 mcg/kg) was then given in a similar fashion, and the same protocol was repeated. These time periods were selected due to the expected pharmacokinetics of norepinephrine and sodium nitroprusside.^27–30

Vasodilator protocol

The sodium nitroprusside used in this protocol was diluted to a concentration of 250 mcg/mL. A constant rate infusion of low-dose norepinephrine ranging from 0.05–0.2 mcg/kg/min was started for a goal mean arterial pressure (MAP) of 30–40 mmHg above baseline to preconstrict the IVC and preserve venous return. Once the goal rate was achieved and the animal’s vitals were stabilized, a repeat set of baseline values were obtained as with the previous protocol. A dose of sodium nitroprusside (10 mcg/kg) was then given as a rapid injection to the abdominal IVC as detailed above, and the same protocol following vasoconstrictor administration was repeated.

Recovery

At the conclusion of the study, all medications were discontinued, and the animal was allowed to return to baseline. The orogastric tube was removed and the sheep was extubated once it was breathing without assistance from the ventilator. The sheep was transferred to a recovery suite for close monitoring. Postprocedural labs, including an arterial blood gas, complete blood count, and basic metabolic panel, were obtained to evaluate for any potential complications.

Data analysis

Data collected for this study included continuous measurements of systolic blood pressure (SBP), diastolic blood pressure (DBP), MAP, heart rate, and respiratory rate obtained from the arterial line and pulse oximeter, which were documented every 5 s, as well as real-time IVC imaging using IVUS (Fig. 1C, D). The degree of vasoconstriction or vasodilation was determined by the percent change in cross-sectional area of the vessel lumen from baseline values. Midgraft/midthoracic IVC pressures and cardiac output were acquired at set time points after medication administration as detailed above. After data collection, arterial line data were visualized graphically over the time course of the experiment to assess the trends in vitals. IVC pressure and cardiac output were overlaid according to their collection time points. IVUS images were analyzed using the method described below. Results following each vasoactive injection were categorized into three intervals consisting of a baseline prior to drug injection, an early phase within 1 minute after drug injection, and recovery phase occurring 3–5 min after drug injection.

VIVUS: an image processing package for venous IVUS data

VIVUS is a Python-based image processing package designed to analyze venous IVUS images with a focus on detecting and quantifying stenosis. The code supports various image formats, including DICOM, PNG, and JPEG, to process cross-sectional views of veins, assess vessel morphology, and compute critical dimensional metrics. For images that depict thin-walled venous structures, the library identifies and returns the contour of the lumen. In cases where stenosis is detected, VIVUS returns contours for both the lumen and the scaffold, highlighting the intimal hyperplasia of constricted portions. This dual-contour capability is necessary to determine the degree of stenosis. Minimum and maximum diameters were calculated to characterize the shape and size of the vessel lumen. Additionally, the equivalent radius was derived from the enclosed area, offering a standardized measure of the vessel size. The perimeter of the contour was analyzed as well to gain further understanding of the vessel structure.

The VIVUS package relies on two deep neural network instance segmentation models:³¹ one to segment the lumen and another to identify stenosis. Both models were based on the Mask- Region-based Convolutional Neural Network (R-CNN) architecture,³² a highly effective framework for instance segmentation tasks that has been used in various modalities of medical image processing. The transfer learning approach on Mask R-CNN enables precise detection and segmentation of regions of interest, making it particularly suitable for differentiating between normal and stenotic areas in IVUS images. These models were trained on a custom dataset acquired from the Phillips Volcano system, consisting of 150 venous IVUS images, each labeled with lumen and stenosis contours when applicable. These labels were manually created and verified by researchers, ensuring quality and accuracy of the training data. Training was conducted using a Google Collab instance with Tesla K80 GPU.

Upon completion of segmentation, VIVUS used a decision tree model to assess whether stenosis was present. This decision tree analyzes the outputs of the two segmentation networks—lumen and stenosis contours—and applies criteria based on area reduction and other geometric features. If stenosis is confirmed, the model computes additional metrics, such as the stenosis percentage. VIVUS has been tested against manual annotations, achieving a Mean Absolute Error (MAE) of approximately 7% in terms of area calculation. By fully automating contouring tasks, VIVUS significantly reduces the time required for image interpretation while ensuring reproducibility.

Results

The vasoreactivity protocol was performed in three sheep with TEVG interposition graft implantations and one aged-matched control with native IVC. A common complication observed following this procedure was acute kidney injury, which was identified on routine lab work obtained the day after as an elevation of creatinine above baseline. This was mild or stage 1 (1.5–1.9 times baseline creatinine) in all animals and improved within 24–48 h with appropriate intravenous fluid administration. No other major complications were observed.

Vital parameters

Overall trends in vitals were similar across the four sheep (specific values can be obtained in Supplementary Table S1). In all animals, heart rate rapidly increased from baseline after both low- and high-dose NE injections, followed by a slow return to baseline during the recovery phase (Fig. 2A, B). Peak heart rate among TEVG sheep occurred at 20 ± 5 and 38 ± 16 s following low and high doses of NE, respectively, and at 30 and 35 s in the control sheep. As expected, a greater increase in heart rate was observed following the high-dose injection of NE compared to the low dose (Supplementary Table S1). The baseline heart rate on a NE continuous infusion was notably lower than baseline heart rates without drug intervention (Fig. 2C). Administration of a SNP injection led to increased heart rate in the early period, which then remained above baseline throughout the recovery.

FIG. 2.

Dynamic changes in vitals during vasoreactivity testing. Hemodynamic responses were assessed in three cases with a tissue-engineered vascular graft implanted as an IVC interposition graft. Heart rate (bpm) is shown in the top (ABC), mean arterial pressure (MAP, mmHg) is in the middle (DEF), and cardiac output (L/min) is in the bottom row (GHI). The left column represents responses to low-dose norepinephrine (ADG), the middle column represents responses to high-dose norepinephrine (BEH), and the right column represents responses to sodium nitroprusside (CFI). Each figure contains five lines: three individual TEVG cases, one average line of all TEVG cases, and one line of the Native case. The black and gray lines represent each individual TEVG, while the bold red line represents the averaged TEVG response. The native IVC control is shown in blue.

Similarly, MAP increased early after both NE injections, then gradually returned towards baseline in recovery (Fig. 2D, E). There was a greater increase in MAP, as well as a slower recovery to baseline, following the administration of high-dose NE compared to the low dose. Peak MAP among TEVG sheep was observed 30 ± 15 s after low-dose NE injection and 40 ± 9 s after high-dose NE injection. In the control sheep, MAP peaked at 35 and 60 s after NE injection. Continuous NE infusion caused a consistent increase in MAP. Administration of SNP then caused an abrupt drop in MAP, followed by a gradual return to baseline during the recovery period (Fig. 2F). The blood pressure nadir occurred 85 ± 23 s after SNP injection in TEVG sheep and at 120 s in the control sheep.

Cardiac output

Cardiac output increased in 2 out of the 3 TEVG sheep during the early time points after low-dose NE injection, with the third having a less robust response to the injection (Fig. 2G). All TEVG sheep then experienced a return to near baseline levels of cardiac output in recovery. With high-dose NE, all TEVG sheep responded with an increase in cardiac output shortly after drug administration, which then also returned to baseline numbers in recovery (Fig. 2H). In contrast, cardiac output initially decreased after low-dose NE injection in the control sheep, though it increased with administration high-dose NE (Fig. 2G, H). Modest changes in cardiac output were observed following SNP injection, with all animals experiencing a slight increase in the early phase (Fig. 2I).

Vasoreactivity (luminal area change)

Changes in the cross-sectional area of the vessel lumen could be grossly identified during the procedure on IVUS imaging (Fig. 3). Following low-dose NE injection, the luminal area of the TEVG midgraft decreased by 16.6 ± 12.8%. Maximum constriction was seen on average 22 ± 3 s after drug administration. The luminal area subsequently increased by 19.1 ± 19.3% compared to baseline during the recovery phase (Fig. 4A, B). A similar pattern was observed on injection of high-dose NE, with a decrease in TEVG luminal area by 15.4 ± 7.0% and an average time to maximum constriction of 22 ± 6 s. This was followed by a 12.3 ± 9.0% increase in luminal area during recovery (Fig. 4C, D). In the control sheep, luminal area of the native IVC decreased in a dose-dependent manner by 16.6% and 40.3% with maximum constriction occurring 27 and 26 s after low- and high-dose NE injection, respectively. Luminal area of the native IVC then increased by 12.3% and 15.1% in the recovery phase (Fig. 4A–D). Notably, administration of SNP resulted in trivial changes to TEVG size, with a 0.7 ± 0.2% increase in luminal area during in the early phase, followed by a 17.8 ± 14.5% decrease from baseline in recovery. A similar effect was seen in the native IVC, with a 0.4% increase in luminal area during the early phase after drug injection, followed by a 14.7% decrease in recovery (Fig. 4E, F).

FIG. 3.

Intravascular ultrasound imaging of dynamic vascular responses to vasoactive agents. IVUS images were captured in three cases with a tissue-engineered vascular graft implanted as an IVC interposition graft. (A) Low-dose norepinephrine, (B) High-dose norepinephrine, and (C) Sodium nitroprusside. Each set contains cross-sectional images taken at three time points: baseline (preinjection), peak response, and recovery phase. Yellow dashed lines indicate the vessel lumen. Below each set of cross-sectional images is a longitudinal IVUS strip showing continuous lumen changes over the full 5-min duration of drug injection. Arrows mark the approximate time of drug injection. Changes in luminal diameter can be observed dynamically along the strip as the drug effects progress and subside.

FIG. 4.

Dynamic changes in vessel area following vasoactive drug injection. Dynamic changes in the vessel cross-sectional area were tracked over time. The baseline cross-sectional area before drug administration was set to 0% for normalization. (A) Low-dose norepinephrine, (B) High-dose norepinephrine, and (C) Sodium nitroprusside. Black to gray lines represent individual TEVG responses, while the red line represents the averaged response of all TEVGs, and the blue line represents the response of the native IVC control. The middle panels display intravascular ultrasound (IVUS) images corresponding to baseline, peak reaction, and recovery phases. The right panels present bar graphs of the peak percent change in TEVG cross-sectional area compared to the baseline for low-dose norepinephrine (D), high-dose norepinephrine (E), and sodium nitroprusside (F). The native IVC control cross-sectional area change is represented as a distinct point in blue.

Midgraft intraluminal pressure

Following low-dose NE injection, TEVG midgraft pressure increased from a baseline value of 2.3 ± 1.2 mmHg to 4.3 ± 1.7 mmHg during the early phase and remained elevated at 4.0 ± 1.6 mmHg throughout the recovery period. Similarly, high-dose NE caused a rise in intraluminal pressure from 3.0 ± 1.4 mmHg to 4.0 ± 1.4 mmHg, with a further increase to 4.8 ± 2.3 mmHg during recovery. In contrast, SNP injection precipitated a drop in pressure, with a decrease from 4.0 ± 2.8 mmHg to 2.7 ± 2.1 mmHg during the early phase and to 2.4 ± 1.7 mmHg during recovery.

Following low-dose NE injection, control sheep IVC pressure increased from a baseline value of 3 mmHg to 5 mmHg during the early phase and remained elevated at 6 ± 0 mmHg throughout the recovery period. Similarly, high-dose NE caused a rise in intraluminal pressure from 6 mmHg to 7 mmHg, with a further increase to 8.3 ± 0.47 mmHg during recovery. In contrast, SNP injection precipitated a drop in pressure, with a decrease from 4 mmHg to 3 mmHg during the early phase and to 2.3 ± 0.47 mmHg during recovery.

Discussion

We developed a protocol to test the physiological response of TEVGs to vasoactive agents in vivo using an ovine IVC interposition graft model. The results demonstrate that neovessels arising from TEVG implants possess the ability to constrict in response to a biochemical stimulus. Overall, the animals’ heart rate and blood pressure responded to vasoactive drug injection as expected. Administration of NE induced rapid and dose-dependent increases in heart rate and MAP, whereas SNP caused a drop in blood pressure associated with the onset of reflexive tachycardia. Among TEVG sheep, cardiac output increased with NE injection in a dose-dependent manner, though limited changes were observed following the administration of SNP, likely due to this compensatory mechanism. In terms of vasoreactivity, both low- and high-dose NE injections produced an immediate and definitive constricting effect on the TEVGs with significant decreases in cross-sectional intraluminal area. Similar results were reproduced within the native IVC of an age-matched control sheep. In contrast, SNP injection did not appear cause any appreciable vasodilation in either TEVGs or native IVC.

Continuous and real-time monitoring of heart rate, blood pressure, and luminal area were necessary to distinguish between active changes occurring in response to a biochemical stimulus from passive changes due to vascular compliance or systemic hemodynamic shifts. In our model, TEVGs reached maximum constriction at the same time or before peaks in heart rate and blood pressure manifested, suggesting a localized, receptor-mediated vasoconstrictive response likely through α1-adrenegic activation prior to full systemic distribution of norepinephrine. This temporal dissociation supports the idea that TEVGs possess functional vasomotor responsiveness, consistent with observations in native venous tissue responding to α-adrenergic agonists in vivo.^33,34 The subsequent paradoxical vasodilation observed during the recovery may reflect the adaption of the IVC acting as a capacitance vessel, accomodating increased venous return driven by peripheral vasoconstriction and augmented cardiac output. This response is consistent with preload-dependent venous dynamics described in previous models.³⁵ Alternatively, delayed compensatory vasodilation may have also been mediated by endogenous responses activated by acute hypertension, such as nitric oxide release or baroreflex-induced sympathetic withdrawal.³⁶ These dynamic changes underscore the importance of assessing TEVG behavior in vivo, where vascular tone is modulated by the interplay of neurohumoral signaling, mechanical forces, and systemic hemodynamic feedback.

Failure to induce a focal response using SNP could be due to several reasons. First, veins have a much thinner vessel wall compared to arteries, with considerably less smooth muscle and elastin content, both of which contribute to their capacity to relax or contract.³⁷ These differences in structure correspond to the divergent roles of arteries, which are responsible for ensuring adequate perfusion throughout the body, and veins, which serve as a functional blood reservoir and facilitate passive blood flow back to the heart. While arteries maintain a basal tone under physiological conditions, the intraluminal area of veins is influenced more greatly by their compliance and the overall blood volume. Additionally, sheep have a natural narrowing of the IVC at the diaphragm, which results in a significant pressure gradient between the thoracic and abdominal cavities.³⁸ Under sedation, thoracic IVC pressures are typically close to zero. While this low pressure might permit some degree of constriction in the presence of limited vascular smooth muscle, it could, conversely, preclude vessel dilation after SNP injection. For instance, our previous ex vivo testing demonstrated that the IVC did not exhibit active dilation in response to SNP unless it was first brought into a preconstricted state. Rather than generating a clear vasodilatory response, SNP primarily led to a reduction in baseline constriction.¹¹ With in vivo testing, while the use of a continuous NE infusion could establish such a preconstricted state, it also alters systemic flow, ultimately increasing venous return. Given that venous structures are highly responsive to fluid volume and intraluminal pressure, the overall effect might have masked attempts to induce localized vasodilation. Lastly, SNP may not be sufficiently fast acting to produce a meaningful response within the short vessel segment under evaluation before it is washed away and the ensuing systemic effects predominated.

Nevertheless, results from this study have significant implications to the clinical translation of TEVGs, particularly in the context of congenital heart diseases, such as single ventricle, requiring venous reconstruction. Vasoreactivity testing in vivo demonstrated that TEVGs are capable of responding to vasoactive agents, indicating that these grafts may play an active role in venous flow dynamics, and consequently, systemic hemodynamics. By modulating their luminal diameter in response to physiological stimuli, TEVGs may be capable of optimizing venous return and improving flow patterns. This adaptation could be particularly beneficial in conditions where controlled venous flow is crucial for maintaining stable central venous pressure and cardiac preload.

These findings are especially pertinent to the Fontan circulation, where long-term organ dysfunction resulting from prolonged elevation in central venous pressures, such as congestive hepatopathy, have been increasingly recognized. The vasoreactive properties of TEVG conduits could mitigate these complications by improving venous flow and alleviating the adverse effects arising from chronic venous hypertension. Moreover, the ability of TEVGs to vasoconstrict or dilate in cohesion with surrounding native vasculature may help to regulate local shear stress and preserve an optimal shear environment, which is important for endothelial function and vascular remodeling, further reducing the risk of venous congestion.

Limitations

Despite promising results, there are limitations to this protocol inherent to the goal of evaluating vasoreactivity in vivo. Notably, there were deviations from preceding results obtained from in vitro experiments.¹¹ However, this was not unexpected, as it is well-documented that results of in vivo vasoreactivity testing differ depending on the animal model and type of vasculature under examination. While NE and SNP were chosen among several other drugs based on their rapid onset and short-acting half-lives, as well as clinical utility, their effects were less predictable in vivo given the inability to control all physiological parameters, such as volume status, endogenous hormone levels, autonomic activity, metabolic factors, and venous flow rate, which can affect exposure time to the vasoactive agent. Furthermore, as the venous system is a high capacitance system, significant differences in fluid status among animals may have contributed to the variance observed in our study results. As fluid status may be impacted by both the individual animal’s behavior and differences in case length, maintenance fluids were provided throughout the procedure. Lastly, small sample size was a primary limitation of this study, especially when trying to capture subtle differences in vasoreactivity. While our current protocol may allow for the identification of major effects and trends, the study lacks the statistical power to detect significant differences between experimental groups. Nevertheless, this experience adds another useful experimental design to the small body of existing literature on in vivo vasoreactivity testing in large animal models. Future studies with larger cohorts will be necessary to validate these initial observations and compare the vasoactive functionality of TEVGs with native IVC, as well as current gold standard synthetic grafts for large vessel conduits. In addition, future histological analysis of TEVG explants will be important to determine neovessel wall composition in comparison to native IVC and identify any dissimilarities that may correlate with differences in functional behavior.

Authors’ Contributions

M.G., D.V., T.W., and M.W. created the method, cowrote the article and led experiments. A.U., A.M., K.N., S.Y., and S.F.H. assisted with experiments. T.S. and C.B. supervised the experiments.

Disclosure Statement

C.B. and T.S. received grant support from Gunze Limited, Pall Corporation, and Cook Regenetec. The remaining authors declare no competing interests.

Funding Information

This research was supported by the Department of Defense Award Number W81XWH-22-1-0597 in addition to NIH grants: R01 HL163065 awarded to C.B.