Mimicking Clinical Rejection Patterns in a Rat Osteomyocutaneous Flap Model of Vascularized Composite Allotransplantation.

INTRODUCTION: Graft loss in vascularized composite allotransplantation (VCA) is more often associated with vasculopathy and chronic rejection (CR) than acute cellular rejection (ACR). We present a rat osteomyocutaneous flap model using titrated tacrolimus administration that mimics the graft rejection patterns in our clinical hand transplant program. Comparison of outcomes in these models support a role for ischemia reperfusion injury (IRI) and microvascular changes in CR of skin and large-vessel vasculopathy. The potential of the surgical models for investigating mechanisms of rejection and vasculopathy in VCA and treatment interventions is presented. MATERIALS AND METHODS: Four rodent groups were evaluated: syngeneic controls (Group 1), allogeneic transient immunosuppression (Group 2), allogeneic suboptimal immunosuppression (Group 3), and allogeneic standard immunosuppression (Group 4). Animals were monitored for ACR, vasculopathy, and CR of the skin. RESULTS: Transient immunosuppression resulted in severe ACR within 2 wk of tacrolimus discontinuation. Standard immunosuppression resulted in minimal rejection but subclinical microvascular changes, including capillary thrombosis and luminal narrowing in arterioles in the donor skin. Further reduction in tacrolimus dose led to femoral vasculopathy and CR of the skin. Surprisingly, femoral vasculopathy was also observed in the syngeneic control group. CONCLUSIONS: Titration of tacrolimus in the allogeneic VCA model resulted in presentations of rejection and vasculopathy similar to those in patients and suggests vasculopathy starts at the microvascular level. This adjustable experimental model will allow the study of variables and interventions, such as external trauma or complement blockade, that may initiate or mitigate vasculopathy and CR in VCA.

Stromal Vascular Fraction Reverses the Age-Related Impairment in Revascularization following Injury.

Adipose-derived stromal vascular fraction (SVF) has emerged as a potential regenerative therapy, but few studies utilize SVF in a setting of advanced age. Additionally, the specific cell population in SVF providing therapeutic benefit is unknown. We hypothesized that aging would alter the composition of cell populations present in SVF and its ability to promote angiogenesis following injury, a mechanism that is T cell-mediated. SVF isolated from young and old Fischer 344 rats was examined with flow cytometry for cell composition. Mesenteric windows from old rats were isolated following exteriorization-induced (EI) hypoxic injury and intravenous injection of one of four cell therapies: (1) SVF from young or (2) old donors, (3) SVF from old donors depleted of or (4) enriched for T cells. Advancing age increased the SVF T-cell population but reduced revascularization following injury. Both young and aged SVF incorporated throughout the host mesenteric microvessels, but only young SVF significantly increased vascular area following EI. This study highlights the effect of donor age on SVF angiogenic efficacy and demonstrates how the ex vivo mesenteric-window model can be used in conjunction with SVF therapy to investigate its contribution to angiogenesis.

Sulforaphane Does Not Protect Right Ventricular Systolic and Diastolic Functions in Nrf2 Knockout Pulmonary Artery Hypertension Mice.

PURPOSE: Nrf2 is a nuclear transcription factor and plays an important role in the regulation of oxidative stress and inflammation. We recently demonstrated that sulforaphane (SFN) protected mice from developing pulmonary arterial hypertension (PAH) and right ventricular (RV) dysfunction by elevating cardiac Nrf2 expression and function. Here we further investigate Nrf2 dependence for SFN-mediated prevention of PAH and RV dysfunction in an Nrf2 knockout mouse model. METHODS: We used male global Nrf2-knockout mice and male C57/6 J wild type mice in the following groups: Control group received room air and vehicle control; SuHx group received SU5416 and 10% hypoxia for 4 weeks to induce PAH; SuHx+SFN group received both SuHx and sulforaphane, a Nrf2 activator, for 4 weeks. Transthoracic echocardiography was performed to quantify RV function and estimate pulmonary vascular resistance over 4 weeks. PAH was confirmed using invasive RV systolic pressure measurement at 4 weeks. RESULTS: All Nrf2 knockout mice survived the 4-week SuHx induction of PAH. SuHx caused progressive RV diastolic/systolic dysfunction and increased RV systolic pressure. The development of RV diastolic dysfunction occurred earlier in the Nrf2 knockout PAH mice when compared with the wide type PAH mice. SFN partially or completely reversed SuHx-induced RV diastolic/systolic dysfunction and increased RV systolic pressure in wild-type mice, but not in Nrf2 knockout mice. CONCLUSION: Our findings demonstrated the essential role of Nrf2 in SFN-mediated prevention of RV dysfunction and PAH, and increasing Nrf2 activity in patients with PAH may have therapeutic potential.

Cell therapy rescues aging-induced beta-1 adrenergic receptor and GRK2 dysfunction in the coronary microcirculation.

Our past study showed that coronary arterioles isolated from adipose-derived stromal vascular fraction (SVF)-treated rats showed amelioration of the age-related decrease in vasodilation to beta-adrenergic receptor (ß-AR) agonist and improved ß-AR-dependent coronary flow and microvascular function in a model of advanced age. We hypothesized that intravenously (i.v.) injected SVF improves coronary microvascular function in aged rats by re-establishing the equilibrium of the negative regulators of the internal adrenergic signaling cascade, G-protein receptor kinase 2 (GRK2) and G-alpha inhibitory (Gαi) proteins, back to youthful levels. Female Fischer-344 rats aged young (3 months, n = 24), old (24 months, n = 26), and old animals that received 1 × 107 green fluorescent protein (GFP+) SVF cells (O + SVF, n = 11) 4 weeks prior to sacrifice were utilized. Overnight urine was collected prior to sacrifice for catecholamine measurements. Cardiac samples were used for western blotting while coronary arterioles were isolated for pressure myography studies, immunofluorescence staining, and RNA sequencing. Coronary microvascular levels of the ß1 adrenergic receptor are decreased with advancing age, but this decreased expression was rescued by SVF treatment. Aging led to a decrease in phosphorylated GRK2 in cardiomyocytes vs. young control with restoration of phosphorylation status by SVF. In vessels, there was no change in genetic transcription (RNAseq) or protein expression (immunofluorescence); however, inhibition of GRK2 (paroxetine) led to improved vasodilation to norepinephrine in the old control (OC) and O + SVF, indicating greater GRK2 functional inhibition of ß1-AR in aging. SVF works to improve adrenergic-mediated vasodilation by restoring the ß1-AR population and mitigating signal cascade inhibitors to improve vasodilation.

Evolution of the rat hind limb transplant as an experimental model of vascularized composite allotransplantation: Approaches and advantages.

As clinical experience with surgical techniques and immunosuppression in vascularized composite allotransplantation recipients has accumulated, vascularized composite allotransplantation for hand and face have become standard of care in some countries for select patients who have experienced catastrophic tissue loss. Experience to date suggests that clinical vascularized composite allotransplantation grafts undergo the same processes of allograft rejection as solid organ grafts. Nonetheless, there are some distinct differences, especially with respect to the immunologic influence of the skin and how the graft is affected by environmental and traumatic insults. Understanding the mechanisms around these similarities and differences has the potential to not only improve vascularized composite allotransplantation outcomes but also outcomes for all types of transplants and to contribute to our understanding of how complex systems of immunity and function work together. A distinct disadvantage in the study of upper extremity vascularized composite allotransplantation recipients is the low number of clinical transplants performed each year. As upper extremity transplantation is a quality of life rather than a lifesaving transplant, these numbers are not likely to increase significantly until the risks of systemic immunosuppression can be reduced. As such, experimental models of vascularized composite allotransplantation are essential to test hypotheses regarding unique characteristics of graft rejection and acceptance of vascularized composite allotransplantation allografts. Rat hind limb vascularized composite allotransplantation models have been widely used to address these questions and provide essential proof-of-concept findings which can then be extended to other experimental models, including mice and large animal models, as new concepts are translated to the clinic. Here, we review the large body of rat hind limb vascularized composite allotransplantation models in the literature, with a focus on the various surgical models that have been developed, contrasting the characteristics of the specific model and how they have been applied. We hope that this review will assist other researchers in choosing the most appropriate rat hind limb transplantation model for their scientific interests.

Thrombospondin-1 mediates Drp-1 signaling following ischemia reperfusion in the aging heart.

BACKGROUND: Ischemia reperfusion (IR) injury leads to activation of dynamin-related protein (Drp-1), causing mitochondrial fission and generation of reactive oxygen species (ROS), but the molecular mechanisms that activate Drp-1 are not known. The purpose of this study was to establish a link between Thbs-1 and fission protein (Drp-1) through Pgc-1α following IR in advancing age. METHODS: Female Fischer-344 rats were divided into four groups: Young Control, Young + IR, Old Control, and Old + IR. Heart function and coronary flow were evaluated at baseline and 72 hours after IR, hearts were explanted and mitochondrial ROS generation was measured using MitoPY1, as well as protein levels of Thbs-1, Pgc-1α, and Drp-1. In vitro, rat aortic endothelial cells (RAEC) were treated with siRNA or plasmid for Pgc-1α to evaluate Pgc-1α effect on Drp-1. RESULTS: Mitochondrial ROS generation in heart tissue increased in both age groups following IR. Old animals exhibited diastolic dysfunction at baseline; after IR they displayed reduced systolic function and exacerbated diastolic dysfunction compared to young controls. IR increased Thbs-1 and Drp-1 expression in young and old hearts compared to control. siRNA to Pgc-1α enhanced levels of Drp-1 in RAECs and increased ROS generation after hypoxia, while Pgc-1α plasmid ameliorates Drp-1 expression in the presence of exogenous Thbs-1. CONCLUSION: These results highlight a novel signaling pathway by which Thbs-1 regulates mitochondrial fission protein (Drp-1) and ROS generation during hypoxia, and presumably, following IR. Inhibiting Thbs-1 immediately after IR may prevent Drp-1-mediated mitochondrial fission and is likely to improve the diastolic function of the heart by reducing ROS-mediated cardiomyocyte damage in the aged population.

Cardiac tissue remodeling in healthy aging: the road to pathology.

This review aims to highlight the normal physiological remodeling that occurs in healthy aging hearts, including changes that occur in contractility, conduction, valve function, large and small coronary vessels, and the extracellular matrix. These "normal" age-related changes serve as the foundation that supports decreased plasticity and limited ability for tissue remodeling during pathophysiological states such as myocardial ischemia and heart failure. This review will identify populations at greater risk for poor tissue remodeling in advanced age along with present and future therapeutic strategies that may ameliorate dysfunctional tissue remodeling in aging hearts.

Adipose-resident myeloid-derived suppressor cells modulate immune cell homeostasis in healthy mice.

The metabolically dynamic nature of healthy adipose places this tissue under regular inflammatory stress. A network of adipose-resident anti-inflammatory immune cells modulates and resolves this endogenous inflammation. Previous work in our laboratory identified a CD11b+ Gr1+ subset of these immunosuppressive adipose stromal cells in healthy mice. Myeloid-derived suppressor cells (MDSCs), typically associated with cancer and chronic inflammation, have a similar surface marker phenotype and accumulate in adipose of high-fat diet-fed mice. Given the routine inflammatory stresses on healthy adipose and the suppressive nature of the tissue-resident immune cells, we hypothesized that these CD11b+ Gr1+ cells were a genuine population of MDSCs involved in regulating tissue homeostasis. Flow cytometric analysis of these cells found that they were CD11b+ CD301- Ly6C+ Ly6G+/- and did not express traditional macrophage markers. Moreover, in vitro functional assays demonstrated that these cells suppressed αCD3/αCD28-induced T-cell proliferation, solidifying their identity as bona fide adipose-resident MDSCs. Systemic MDSC depletion altered adipose immune cell dynamics in otherwise healthy mice, increasing the number of CD4+ effector memory T cells and modifying the surface markers expressed by adipose-resident macrophages. In addition, transcription of various immunomodulatory cytokines was clearly dysregulated in the adipose of MDSC-depleted animals compared with controls. Altogether, our findings indicate that there is a population of bona fide MDSCs in the adipose of otherwise healthy mice that actively contribute to the health and immune homeostasis of this tissue.

Enhanced beta-1 adrenergic receptor responsiveness in coronary arterioles following intravenous stromal vascular fraction therapy in aged rats.

Our past study showed that a single tail vein injection of adipose-derived stromal vascular fraction (SVF) into old rats was associated with improved dobutamine-mediated coronary flow reserve. We hypothesize that i.v. injection of SVF improves coronary microvascular function in aged rats via alterations in beta adrenergic microvascular signaling. Female Fischer-344 rats aged young (3 months, n=32) and old (24 months, n=30) were utilized, along with two cell therapies intravenously injected in old rats four weeks prior to sacrifice: 1x107 green fluorescent protein (GFP+) SVF cells (O+SVF, n=21), and 5x106 GFP+ bone-marrow mesenchymal stromal cells (O+BM, n=6), both harvested from young donors. Cardiac ultrasound and pressure-volume measurements were obtained, and coronary arterioles were isolated from each group for microvessel reactivity studies and immunofluorescence staining. Coronary flow reserve decreased with advancing age, but this effect was rescued by the SVF treatment in the O+SVF group. Echocardiography showed an age-related diastolic dysfunction that was improved with SVF to a greater extent than with BM treatment. Coronary arterioles isolated from SVF-treated rats showed amelioration of the age-related decrease in vasodilation to a non-selective ß-AR agonist. I.v. injected SVF cells improved ß-adrenergic receptor-dependent coronary flow and microvascular function in a model of advanced age.

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR). In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.