Transplant Vascular Disease Research Articles

Cerebrovascular Disease Hospitalization Rates in End-Stage Kidney Disease Patients with Kidney Transplant and Peripheral Vascular Disease: Analysis Using the National Inpatient Sample (2005-2019).

Individuals with end-stage kidney disease (ESKD) face higher cerebrovascular risk. Yet, the impact of peripheral vascular disease (PVD) and kidney transplantation (KTx) on hospitalization rates for cerebral infarction and hemorrhage remains underexplored. Analyzing 2,713,194 ESKD hospitalizations (2005-2019) using the National Inpatient Sample, we investigated hospitalization rates for ischemic and hemorrhagic cerebrovascular diseases concerning ESKD, PVD, KTx, or their combinations. Patients hospitalized with cerebral infarction due to thrombosis/embolism/occlusion (CITO) or artery occlusion resulting in cerebral ischemia (AOSI) had higher rates of comorbid ESKD and PVD (4.17% and 7.29%, respectively) versus non-CITO or AOSI hospitalizations (2.34%, p < 0.001; 2.29%, p < 0.001). Conversely, patients hospitalized with nontraumatic intracranial hemorrhage (NIH) had significantly lower rates of ESKD and PVD (1.64%) compared to non-NIH hospitalizations (2.34%, p < 0.001). Furthermore, hospitalizations for CITO or AOSI exhibited higher rates of KTx and PVD (0.17%, 0.09%, respectively) compared to non-CITO or AOSI hospitalizations (0.05%, p = 0.033; 0.05%, p = 0.002). Patients hospitalized with NIH showed similar rates of KTx and PVD (0.04%) versus non-NIH hospitalizations (0.05%, p = 0.34). This nationwide analysis reveals that PVD in ESKD patients is associated with increased hospitalization rates with cerebral ischemic events and reduced NIH events. Among KTx recipients, PVD correlated with increased hospitalizations for ischemic events, without affecting NIH. This highlights management concerns for patients with KTx and PVD.

GW24-e2442 Wnt3a activates β1-integrin and regulates migration and adhesion of vascular smooth muscle cells

ObjectivesVascular smooth muscle cells (VSMCs) are known to undergo functional changes that can contribute to the pathogenesis of atherosclerosis and restenosis. Wnts are a family of secreted glycoproteins that bind...

Soluble donor-like MHC class I proteins induce CD4 +CD25 +CD8 −FoxP3 + cells with potential to ameliorate graft chronic injury

Conventional immunosuppressive therapies failed to prevent allograft chronic rejection. New approaches to modulate recipient immune response are needed. Donor-like MHC class I soluble proteins demonstrated therapeutic potential to suppress chronic rejection. The present study was designed to clarify the ability of MHC class I soluble proteins to induce T regulatory cells with true regulatory potential in a fully allogeneic rat cardiac transplant model. Donor-like MHC class I proteins upregulate small population of splenic CD8 − negative CD4 +CD25 +FoxP3 + positive cells. CD4 + splenocytes after MHC therapy suppress lymphocyte proliferation against donor antigens in vitro. ACI recipients of WF hearts treated with CD4 + cells, induced with donor-like MHC class I proteins (CD4-MHC), demonstrated stable survival of the transplanted organ (MST > 120 days; n = 17). Histology revealed that grafts of recipients treated with CD4-MHC had 23.6% vessels affected 100 days postgrafting. On the contrary, hearts obtained from long-term surviving hosts treated with CD4 + cells induced with high-dose CsA (CD4-CsA) had 50–70% of affected vessels. CD4-MHC class I treated transplants were mostly CD3 − negative, had low level of mast and FoxP3 + cell infiltration compared to CD4-CsA treated hearts. Intragraft CD4 + cells were close to mast cells in morphology. The same graft tissues had similar number of CD4 + positive cells and mast cells suggesting existence of CD4 + positive mast cells. On the other hand, a negligible number of FoxP3 + positive cells in the grafts after CD4-MHC treatment supports the idea of CD4 + positive FoxP3 + negative mast cells population. We demonstrate that donor-like MHC class I protein therapy induces population of CD4 +CD25 +CD8 −FoxP3 + cells with potential to ameliorate development of transplant vascular disease and evoke CD4 + positive FoxP3 negative mast cells in the secondary hosts.

Granzyme B Induces Endothelial Cell Apoptosis and Contributes to the Development of Transplant Vascular Disease

Endothelial cell death induced by cytotoxic T cells is a key initiating event in the development of transplant vascular disease (TVD), the leading cause of late solid organ transplant failure. We studied the role of the granzyme B (GrB) pathwaye, which is one of the main mechanisms by which T cells induce apoptosis of allogeneic targets, in the pathogenesis of TVD. Granzyme B, in combination with perforin (pfn), induced apoptosis of cultured endothelial cells. In hearts transplanted into GrB knockout (GrB-KO) mice, there was a similar level of vasculitis as compared to WT mice, indicating that GrB does not affect immune infiltration into allograft arteries. However, there was a significant reduction in luminal narrowing of allograft arteries from GrB-KO mice as compared to WT recipients. These results indicate that GrB plays a role in endothelial cell death in allograft arteries and in the resultant development of TVD.

Cardiac transplantation and resistance artery myogenic tone

Transplantation is an effective treatment for end-stage heart disease; however, most grafts eventually fail by progressive cardiac failure. Primarily, failure is ischemic due to the occlusive nature of transplant vascular disease (TVD). Early after transplantation and preceding TVD, alterations in coronary physiology such as reduced vascular myogenic tone occur. Resistance arteries possess an inherent ability to constrict in response to transmural pressure; this constrictive response (myogenic tone) is important in fluid homeostasis. Recent evidence suggests that a decline in myogenic tone leads to deficits in cardiac contractility. Factors that reduce myogenic tone in transplantation include constitutive nitric oxide synthase and inducible nitric oxide synthase catalyzed, NO-mediated vasodilation as well as deficits in arterial contractile function. Reduced myogenic tone in allograft resistance arteries increases coronary blood flow such that hydrostatic pressure surpasses oncotic pressure, causing cardiac interstitial edema. This generalized edema decreases ventricular compliance leading to heart failure during the course of acute immune rejection of the graft. Cyclosporine A treatment reduces immune mediated dysregulation of myogenic tone, resulting in reduced interstitial edema and improved cardiac function. In this review, we discuss aspects of TVD and myogenic tone signaling mechanisms and how aberrations in myogenic regulation of arterial tone contribute to functional changes observed in cardiac transplant.

Perforin Mediates Endothelial Cell Death and Resultant Transplant Vascular Disease in Cardiac Allografts

T cell-induced endothelial injury is an important event in the development of transplant vascular disease (TVD), the leading expression of chronic rejection of vascularized organ transplants. However, the precise contribution of perforin to vascular damage in allografts and resultant TVD has not been addressed in vivo. Minor histocompatability antigen mismatched mouse heterotopic cardiac transplants were performed from 129J donors into C57Bl/6 (wild-type (WT)) or perforin knockout (PKO) recipients. Perforin was abundant in immune infiltrates in the myocardium and vasculature of transplanted hearts in WT mice. Allograft coronary arteries in both WT and PKO mice had considerable vasculitis. There was also marked endothelial disruption, as well as TUNEL-positivity in the endothelial region, in coronary arteries of hearts transplanted into WT mice that was not evident in PKO recipients (P = 0.05). At 30 days post-transplantation, intimal thickening was assessed on elastic Van Gieson-stained ventricular sections. There was an average of 54.2 +/- 6.7% luminal narrowing of coronary arteries in allografts from WT mice as compared to 13.4 +/- 5.1% luminal narrowing in PKO counterparts (P < 0.00002). In summary, perforin plays a primary role in endothelial damage and the resultant onset and progression of TVD.

Studies of LDL particle size and susceptibility to oxidation and association with glucose metabolism in children after heart transplantation

Background Increased concentrations of serum triglyceride and low-density lipoprotein (LDL) cholesterol are common after heart transplantation (HTx). These abnormalities may promote transplant vascular disease and atherosclerosis, especially if LDL is small, dense, and oxidized. There have been no previous studies of LDL particle size and LDL susceptibility to oxidation in children after HTx. Methods Twenty-three HTx recipients (aged 3–19 years) who received triple-drug immunosuppression therapy after HTx and 181 controls within the same age range participated in the study. Total, high-density lipoprotein, and LDL-cholesterol concentrations; triglyceride concentration, and glucose and insulin concentrations during oral glucose tolerance tests were determined an average of 3 years after HTx (range, 1–7 years). Moreover, we determined serum lipoprotein (a) concentration, apolipoprotein E phenotype, LDL particle size, and indices of LDL susceptibility to copper-induced oxidation in 12 HTx recipients. Results We found hypertriglyceridemia in 56.5% and hyperinsulinemia in 30.4% of patients. Triglyceride concentration and body mass index were associated significantly with insulin concentration ( p < 0.008 for both). Low-density lipoprotein particle size, LDL susceptibility to in vitro oxidation, and lipoprotein (a) concentrations did not differ significantly between HTx patients and controls. Low-density lipoprotein particle size was associated inversely with cyclosporine through level (Neoral, r = –0.59, p = 0.045), whereas weight-adjusted dosage of cyclosporine correlated positively with longer lag time of LDL oxidation ( r = 0.69, p = 0.013). Conclusions Hypertriglyceridemia and hyperinsulinemia were common in children receiving triple-drug immunosuppression therapy after HTx. Increased cyclosporine through concentration was associated with small LDL particle size but did not increase LDL susceptibility to oxidation.

Ultrastructural evidence of early endothelial damage in coronary arteries of rat cardiac allografts

Background: Events that occur early after transplantation, particularly immune recognition of allo-endothelium, initiate transplant vascular disease (TVD). Previous work suggests an important compromise of endothelial integrity as the allo-immune milieu evolves, although mechanisms by which integrity is altered remain unclear. Increased vascular permeability caused by endothelial damage may allow inflammatory cells, lipoproteins, other proteins, and plasma fluid to enter the sub-endothelial space, thereby contributing to the initiation of atherosclerosis. In this study, we examined endothelial integrity in coronary arteries and the proximal aorta after cardiac transplantation in rats. Methods: We used Lewis-to-Lewis and Lewis-to-F344 rat heterotopic cardiac transplant models. We studied the effects of cyclosporine (5mg/kg/day) therapy compared with saline-treated controls. En face silver nitrate staining was performed to demonstrate endothelial cell borders and gaps. We used scanning electron microscopy to extend silver nitrate findings and to further define the presence and nature of endothelial disruptions. We used transmission electron microscopy to further characterize immune cell identity and interaction with endothelium. Results: Syngrafts and cyclosporine-treated allografts showed normal-looking endothelium similar to that observed in arteries from native hearts. However, saline-treated allografts displayed progressive endothelial destruction, including large intercellular gaps, missing cells, and areas of bare extracellular matrix. Exfoliated surfaces were covered by platelets at various stages of adhesion, activation, and spreading. Similarly, we observed numerous leukocytes as either adherent to the endothelial lining or transmigrating into the sub-endothelial space. Cessation of cyclosporine therapy was associated with the development of similar abnormalities. Conclusions: Our findings indicate that, especially when immunosuppression is insufficient, early endothelial damage may promote vascular permeability and thereby initiate TVD.

Granzyme B in Atherosclerosis and Transplant Vascular Disease: Association with Cell Death and Atherosclerotic Disease Severity

Apoptosis of intimal cells is an important contributor to the pathogenesis of atherosclerosis and transplant vascular disease (TVD). Since the activated immune response may be a key regulator of apoptosis in these lesions, we used immunohistochemistry to characterize the presence and localization of granzyme B, a major mediator of the cytotoxic immune response, in advanced atherosclerosis and TVD. Formalin-fixed, paraffin-embedded transverse sections from human left anterior descending coronary arteries were cut serially and stained with antibodies specific for granzyme B, smooth muscle α-actin, CD68, and CD3. The amount of granzyme B staining was semi-quantitated on a 0–5+/5+ scale. Also, TUNEL staining and in situ hybridization was performed to visualize cells undergoing cellular damage suggestive of apoptosis, and to localize granzyme B mRNA, respectively. Granzyme B localization was similar in both diseases. This protease was absent in arteries with mild atherosclerosis, but was abundant in the intima and media of vessels with advanced atherosclerosis and TVD. Within the intima, granzyme B localized to TUNEL-positive foam cells surrounding lipid-rich atheromas. Staining of serial sections with granzyme B and either smooth muscle α-actin, anti-CD68, or anti-CD3 showed that granzyme B localized to smooth muscle cells, macrophages, and T-cells. Further, in situ hybridization for granzyme B mRNA in TVD cases localized its expression to infiltrating leukocytes and not foam cells. In conclusion, the presence of granzyme B in advanced atherosclerotic lesions and TVD is associated with increasing disease severity and cell death. These observations suggest that granzyme B-mediated apoptosis may contribute to the pathogenesis of these diseases.

Cardiac allograft vasculopathy and dysregulation of the NO synthase pathway.

Cardiac allograft vasculopathy is the most aggressive form of atherosclerosis in humans and is the leading cause of death after the first year of heart transplantation. Endothelial dysfunction is a major contributing factor to the acceleration of coronary vascular disease in these individuals. A reflection of this endothelial dysfunction is the severe impairment in endothelium-dependent vasodilation that occurs early after transplantation. The etiology of this allograft endothelial alteration is multifactorial and may include preexisting atherosclerosis of the graft vessels, reperfusion injury during transplantation, denervation, disruption of the lymphatic system, and acute and chronic immune injury, as well as traditional risk factors for coronary artery disease (hyperlipidemia, diabetes, hypertension, or hyperhomocysteinemia) and pathogens, such as cytomegalovirus. The alteration in endothelial function affects vasomotor tone of the coronary arteries. Evidence indicates that there may be an impairment of endothelial production and/or activity of NO. Because NO is a potent vasodilator, its deficiency would explain the abnormal vasomotor tone in these individuals. In addition, because NO inhibits key processes in vascular inflammation and atherosclerosis, its absence may contribute to the acceleration of transplant vascular disease. Recent studies from our group and others have shed light on the mechanisms of endothelial dysfunction and its importance in cardiac allograft vasculopathy. In addition, the alteration in endothelial function contributes to vascular inflammation and progression of the disease.

The regulation and consequences of immune-mediated cell death in atheromatous diseases.

Atheromatous diseases are lipid and cell-rich vascular disorders that include coronary artery disease (CAD), transplant vascular disease (TVD), and restenosis. Considering the inflammatory nature of these diseases, cytotoxic immune mechanisms such as the FasL and granzyme/perforin pathways most likely play important roles in the development and remodeling of many lesions. Furthermore, although the contributions of immune responses to each disease vary, the correspondent localization of certain mediators and effectors suggests that they may contribute to a spectrum of atheromatous diseases. In this review, the contribution of immune cell-mediated cell death in the onset and pathogenesis of CAD and TVD is examined.

Granzyme B, a cytotoxic immune protease, induces apoptosis of smooth muscle cells in the absence of perforin

The granzyme/perforin pathway is a key mediator of the cytotoxic immune response to allogeneic tissue/cells. Granzyme B (GrB), the most potent granzyme, localizes to apoptotic smooth muscle cells in human transplant vascular disease (TVD). In this report we examine the effects of GrB on smooth muscle cells (SMC) in vitro. Human coronary artery SMC were cultured to 70% confluence, serum starved, and treated with varying doses of purified GrB for 24h. Surprisingly, GrB alone induced morphological changes that included cell rounding and surface detachment. MTS assay indicated that GrB alone dose-dependently reduced cell viability. There was a significant reduction in cell viability that began at 1.25μg/mL GrB and reached a minimum of 65% of untreated control at 5μg/mL GrB. Hoechst nuclear staining and Annexin V labeling of SMC treated with GrB (2.5μg/mL) demonstrated that the rounded SMC were apoptotic, and Western analysis indicated that GrB activated both caspase-3 and -8. The addition of z-VAD.fmk (multi-caspase inhibitor) or z-AAD.cmk (GrB inihibitor) to SMC prior to treatment with GrB completely inhibited cell death. Further, addition of fluorscently labeled GrB showed that this protease is internalized by SMC. However, although the addition of 20mM soluble mannose-6-phosphate (M6P) inhibited GrB internalization, it did not inhibit cell death. These novel results suggest that GrB may contribute to the pathogenesis of TVD by inducing apoptosis of SMC, at least in part, through a cytotoxic pathway that is independent of perforin. A potential mechanism could involve proteolysis of extracellular and/or plasmalemmal proteins.

Characterization of fractalkine (CX3CL1) and CX3CR1 in human coronary arteries with native atherosclerosis, diabetes mellitus, and transplant vascular disease

Background: Fractalkine is a novel chemokine that mediates both firm adhesion of leukocytes to the endothelium via CX3CR1 and leukocyte transmigration out of the bloodstream. Fractalkine has recently been shown to play a role in the pathogenesis of acute organ rejection. Since its expression is regulated by inflammatory agents such as LPS, IL-1, and TNF-α, fractalkine involvement in atherosclerosis and transplant vascular disease (TVD) is of particular interest. In this study, we characterized the presence of fractalkine and its receptor CX3CR1 in human coronary arteries from normal, atherosclerotic, diabetic, and TVD settings. Method: Polyclonal rabbit antibodies were used to immunostain human fractalkine and CX3CR1 to localize their presence in transverse sections of the proximal left anterior descending and/or right coronary arteries. Slides were scored in a blinded fashion for intensity of staining (0 to 4+) and for localization in vessel walls. Results: Normal coronary arteries showed no fractalkine staining. In atherosclerotic coronary arteries, staining was localized to the intima, media, and adventitia. Within the media, fractalkine expression was seen in macrophages, foam cells, and smooth muscle cells (SMCs). Diabetic vessels showed similar staining patterns to atherosclerotic coronaries, with much stronger staining in the deep intima. Transplanted coronaries showed staining in the endothelium, intima, and adventitia in early disease, and intimal, medial, and adventitial staining in late disease. CX3CR1 staining was seen in the coronary arteries of all cases, with specific localization to regions with fractalkine staining. Conclusion: The distinctive staining patterns in native atherosclerosis, diabetes mellitus with atherosclerosis, and TVD indicate that the expression of fractalkine and CX3CR1 may be important in the pathogenesis of these diseases.

New targets for the prevention of transplant vascular disease

New targets for the prevention of transplant vascular disease

Vascular endothelial growth factor-D (VEGF-D) is an endothelial hyperpermeability inducing growth factor differentially expressed in human cardiac allografts

Purpose: The pathogenesis of transplant vascular disease (TVD) is only partly elucidated. We hypothesize that VEGF-D contributes to the development of atheromatous lesions in TVD through a hyperpermeability inducing effect on the endothelium.

Transplant vascular disease: Potential sites of intervention

Transplant vascular disease: Potential sites of intervention

An overview of the vascular response to injury: a tribute to the late Russell Ross

The innermost layer of a normal blood vessel wall, the tunica intima, consists of a simple monolayer of endothelial cells seated on a basement membrane. Expansion of the intima is a common characteristic of atherosclerosis, restenosis after angioplasty, late closure of saphenous vein grafts and transplant vascular disease. The intima becomes a complex connective tissue containing vascular smooth muscle cells that have invaded from the underlying tunica media and inflammatory cells that have invaded from the circulation. This brief review will concentrate on the molecular events underlying the generation of this neointima ‘in response to injury’ and its consequences for disease. It will also consider the implications for the consequences and early detection of vascular drug toxicity.

Emerging roles of endothelial cells and smooth muscle cells in transplant vascular disease

The lesion-promoting events and processes involved in the pathogenesis of transplant vascular disease (TVD) are intimately dependent on the interactions of both endothelial cells (EC) and smooth muscle cells (SMC) in the vessel wall of solid organ allografts. Immune-mediated recognition and injury of the endothelial cell layer provide the stimulus for the elaboration of EC-derived cytokines and chemokines, and the upregulation of adhesion molecule expression involved in increased leukocyte recruitment and attachment, altered vascular permeability to lipids and cells, stimulation of SMC migration and proliferation, and activation of apoptosis. T cells and macrophages, subsequent to activation and transendothelial migration, further amplify the concentration of soluble mediators in the allograft microenvironment and, together with activated EC, provide a chemokine gradient leading to SMC migration from the media and subsequent proliferation and extracellular matrix synthesis in the neointimal space. The combination of altered EC permeability and SMC matrix synthesis provides a mechanism for lipoprotein insudation and retention, respectively, in the developing lesion. Such lipid accumulation can lead to oxidative modifications that are potentially proatherogenic. Oxidized lowdensity lipoprotein can contribute to further lipid retention, cell proliferation, foam cell formation, and cell death. Nitric oxide derived from vascular cells is also emerging as an important mediator of atherogenesis and may have both antiatherogenic and proatherogenic capabilities. Thus, the activation, injury, or destruction of ECs and SMCs leads to the expansion of a lipid-, SMC-, and matrix-rich intimai lesion, loss of normal vascular function, and the compromise of allograft viability and performance.

Effect of IL-4 deletion on cardiac allograft survival in the BALB/c to 129Sv x C57BL/6 strain combination.

To investigate the effect of IL-4 deletion on cardiac allograft survival, vascularized BALB/c cardiac allografts were placed in C57BL/6, 129Sv x C57BL/6 (IL-4 +/+) or 129Sv x C57BL/6 IL-4 knockout mice (IL-4-/-). Untreated recipients rejected allografts in < 15 days while isografts survived indefinitely (> 100 days). Treatment with anti-CD4 (GK1.5) for 4 days at the time of allografting increased mean survival to > 100 days in C57BL/6, 90+/-16 days in 129Sv x C57BL/6 (IL-4 +/+) and 68 +/- 36 days in 129Sv x C57BL/6 (IL-4-/-) recipients. Although there was a trend towards shorter survival times in the IL-4-/- mAb-treated mice, survival in the three recipient groups was not significantly different (P = 0.07). A 30-day course of anti-CD4 did not further prolong BALB/c heart survival. All long-surviving hearts had histological evidence of parenchymal damage and transplant vascular disease. None of these recipients developed antigen-specific tolerance, since both donor and third party skin graft challenges were rejected when challenged at > 60 days post-graft and all primary grafts failed by 120 days. Thus the effects of IL-4 deletion were subtle and were seen only with low doses of immunosuppression in this high responder strain combination.

COMBINED THERAPY WITH PRAVASTATIN (PVS) AND CYCLOSPORIN (CS) DECREASES ARTERIAL INTIMAL THICKENING IN A MODEL OF SEVERE VASCULAR IMMUNE INJURY

144 Arterial intimal thickening produced by transplant vascular disease (TVD) is the major complication limiting long-term graft survival after solid organ transplantation. We have shown that PVS-treated heart transplant recipients have decreases in the incidence and progression of TVD. It has been hypothesized that the combination of PVS, an HMGCoA reductase inhibitor, and CS in these patients leads to this reduction in TVD. The purpose of this study is to examine the effects of PVS and CS on arterial intimal thickening in a transplant model. Femoral arteries from Brown-Norway rats were transplanted into Lewis rats. Recipient rats were gavaged with PVS (20mg/kg/d), CS(5mg/kg/d) or a combination of PVS and CS beginning either postoperatively(Grp I) or 3 days preoperatively (Grp II). There were six rats in each treatment arm. The transplanted femoral vessels were harvested at postop day 40 and were examined using morphometric and histopathologic techniques. In Grp I, there were no differences in intimal area (mm2, ± S.E.) comparing PVS, CS or PVS+CS treated rats with controls despite a trend towards less inflammation with CS treatment and sparing of medial necrosis with PVS+CS treatment. In Grp II, there was a synergistic decrease in intimal area in PVS+CS treated rats (p<0.05) compared with controls, CS or PVS alone treated rats (see graph). In addition, in Grp II, CS again showed decreased inflammation and PVS+CS-treated rats had minimal medial necrosis despite significant inflammation. FigureIn conclusion, PVS and CS synergistically inhibit arterial intimal thickening when treatment is begun 3 days prior to transplant. Given the lack of intimal thickening despite the presence of mononuclear inflammation, pretreatment with this combination of agents may prevent the elaboration of growth factors responsible for TVD. These findings may help explain the decreased incidence of TVD noted on our PVS-treated heart transplant patients.