Mahesh Krishnamoorthy

Severe venous neointimal hyperplasia prior to dialysis access surgery

BACKGROUND Venous neointimal hyperplasia is the most common cause of arteriovenous (AV) fistula and graft dysfunction following dialysis access surgery. However, the pathogenetic impact of pre-existing venous neointimal hyperplasia at the time of AV access creation on final clinical success is currently unknown in the setting of advanced chronic kidney disease (CKD) and end-stage renal disease (ESRD) patients. The aim of this study was to perform a detailed histological, morphometric, and immunohistochemical analysis of vein specimens in advanced CKD and ESRD patients collected at the time of new vascular access placement. METHODS Vein samples from 12 patients were collected at the time of AV access creation near the site of AV anastomosis. Histological, immunohistochemistry and morphometric studies were performed on these vein samples. RESULTS Examination of the tissue specimens obtained at the time of surgery showed neointimal hyperplasia in 10 of 12 specimens, ranging from minimal to very severe. The majority of cells within the neointima were myofibroblasts with a minority of contractile smooth muscle cells present. CONCLUSION Our work represents a detailed description of the morphometric and cellular phenotypic lesions present in the veins of CKD and ESRD patients, prior to dialysis access placement. These studies (i) suggest the future possibility of a new predictive marker (pre-existing venous neointimal hyperplasia) for AV dialysis access dysfunction and (ii) open the door for the future development of novel local therapies for optimization of the venous substrate on which the dialysis access is created.

Cellular phenotypes in human stenotic lesions from haemodialysis vascular access

BACKGROUND Haemodialysis vascular access dysfunction (due to venous stenosis and thrombosis) is a leading cause of hospitalization and morbidity. The aim of the current study was to identify the specific cell types present within stenotic tissue samples from patients with AV fistula and graft failure. METHODS Discarded tissue segments were collected from the stenotic portions (usually near the graft-vein anastomosis or the AV anastomosis) of 23 dialysis grafts and 20 AV fistulae, and examined for expression of smooth muscle alpha actin, desmin, vimentin and a macrophage marker. RESULTS The majority of cells within the venous neointima (both grafts and fistulae) were myofibroblasts, with a smaller number of desmin positive smooth muscle cells. The graft neointima had a similar cellular phenotype, albeit without any desmin positive contractile smooth muscle cells. The majority of cells within the PTFE graft material were macrophages. Analysis of sequential sections revealed the presence of fibroblasts within the venous neointima and intragraft region. CONCLUSIONS Our results demonstrate that contractile smooth muscle cells, myofibroblasts, fibroblasts and macrophages all play a role in the pathogenesis of dialysis access dysfunction (grafts and fistulae). Targeting these specific cell types might result in the development of novel therapeutic paradigms for haemodialysis vascular access dysfunction.

Hemodynamic wall shear stress profiles influence the magnitude and pattern of stenosis in a pig AV fistula

Venous stenosis is a significant problem in arteriovenous fistulae, likely due to anatomical configuration and wall shear stress profiles. To identify linkages between wall shear stress and the magnitude and pattern of vascular stenosis, we produced curved and straight fistulae in a pig model. A complete wall stress profile was calculated for the curved configuration and correlated with luminal stenosis. Computer modeling techniques were then used to derive a wall shear stress profile for the straight arteriovenous fistula. Differences in the wall shear stress profile of the curved and straight fistula were then related to histological findings. There was a marked inverse correlation between the magnitude of wall shear stress within different regions of the curved arteriovenous fistula and luminal stenosis in these same regions. There were also significantly greater differences in wall shear stress between the outer and inner walls of the straight as compared to curved arteriovenous fistula, which translated into a more eccentric histological pattern of intima-media thickening. Our results suggest a clear linkage between anatomical configuration, wall shear stress profiles, and the pattern of luminal stenosis and intima-media thickening in a pig model of arteriovenous fistula stenosis. These results suggest that fistula failure could be reduced by using computer modeling prior to surgical placement to alter the anatomical and, consequently, the wall shear stress profiles in an arteriovenous fistula.

Influence of Temporal Variation in Wall Shear Stress on Intima-Media Thickening in Arteriovenous Fistulae

Arteriovenous fistula (AVF) failure is mainly due to venous stenosis characterized by significant amount of intima‐media thickening (IMT), probably in the presence of negative (inward) remodeling. Our hypothesis is that the longitudinal changes in wall shear stress (WSS) within different configurations of AVF can influence remodeling factors (changes in luminal diameter (ΔDh) and IMT) during its maturation process. Dh is an equivalent diameter for a noncircular conduit. A total of six AVFs with curved (C‐AVF; n = 3) and straight (S‐AVF; n = 3) configurations were created between the femoral artery and vein of three pigs, bilaterally. CT scans and ultrasounds were utilized to calculate local WSS at 2D (D: days), 7D, and 28D postsurgery. For each AVF, IMT was measured at four regions along the vein using morphometric analyses. At these regions, repeated measurements of WSS and luminal diameter of each AVF were obtained over time. The ΔDh between 7D and 28D was significantly larger for C‐AVF than for S‐AVF (2.27 ± 0.67 mm vs. 0.02 ± 0.55 mm; p < 0.05). Also, at 28D the amount of IMT in C‐AVF (77.46 ± 7.10 units) was significantly greater (p < 0.05) when compared with S‐AVF (53.71 ± 8.23 units). These structural changes were accompanied by significantly different gradients of WSS over time (τ′) for C‐AVF (−0.56 ± 0.60 dyne/cm2/day) in comparison with S‐AVF (0.71 ± 0.39 dyne/cm2/day). Negative τ′ for C‐AVF corresponded to reduction in WSS level over time resulting in a physiological level of WSS at 28D (4.08 ± 5.08 dyne/cm2). In contrast, a positive τ′ for S‐AVF was associated with the increase in WSS levels over time causing high levels of WSS at 28D (36.68 ± 5.32 dyne/cm2). The decrease in WSS levels for the C‐AVF over time was associated with outward remodeling of the venous wall (favorable to maturation). In contrast, for S‐AVF, the increase in WSS levels over time was associated with inward remodeling and subsequently, venous stenosis. Thus, temporal gradients of WSS, which could be altered by the surgical configuration of AVF, may provide important information on the remodeling behavior of AVFs. Identification of an optimal AVF configuration, which results in a temporal decrease in WSS and an outward remodeling of the venous wall, may reduce AVF maturation failure.

Anatomic configuration affects the flow rate and diameter of porcine arteriovenous fistulae

Although arteriovenous fistulae are currently the preferred form of vascular access, early failure is a significant problem. Since wall shear stress is thought to play an important role in the pathogenesis of early failure, and this stress varies markedly in different fistula configurations, we assessed the influence of configuration (curved or straight) on longitudinal changes of flow rate and lumen diameter in a porcine fistula model. Fistulae were created in eight pigs between the femoral artery and vein, with each animal having a curved and a straight configuration on opposite sides. Velocity measurements were obtained by ultrasound at the time of surgery and at intermediate time points up to 28 days. Quantification of both the configuration and the internal diameter of the fistulae was determined by CT scans. The overall rate of increased flow during each time interval (0 to 2 days, 2 to 7 days, and 7 to 28 days) was more pronounced with the curved fistulae. Moreover, the luminal diameter of curved fistulae had dilated more from the time of surgery to 28 days as compared to the straight fistulae. Thus, anatomical configuration of fistulae plays a major role in flow-mediated dilatation. Identifying the optimal configuration may result in increased diameter and consequently blood flow, and perhaps reduce the incidence of early failure.

Effect of Blood Viscosity on Oxygen Transport in Residual Stenosed Artery Following Angioplasty

The effect of blood viscosity on oxygen transport in a stenosed coronary artery during the postangioplasty scenario is studied. In addition to incorporating varying blood viscosity using different hematocrit (Hct) concentrations, oxygen consumption by the avascular wall and its supply from vasa vasorum, nonlinear oxygen binding capacity of the hemoglobin, and basal to hyperemic flow rate changes are included in the calculation of oxygen transport in both the lumen and the avascular wall. The results of this study show that oxygen transport in the postangioplasty residual stenosed artery is affected by non-Newtonian shear-thinning property of the blood viscosity having variable Hct concentration. As Hct increases from 25% to 65%, the diminished recirculation zone for the increased Hct causes the commencement of pO(2) decrease to shift radially outward by approximately 20% from the center of the artery for the basal flow, but by approximately 10% for the hyperemic flow at the end of the diverging section. Oxygen concentration increases from a minimum value at the core of the recirculation zone to over 90 mm Hg before the lumen-wall interface at the diverging section for the hyperemic flow, which is attributed to increased shear rate and thinner lumen boundary layer for the hyperemic flow, and below 90 mm Hg for the basal flow. As Hct increases from 25% to 65%, the average of pO(2,min) beyond the diverging section drops by approximately 25% for the basal flow, whereas it increases by approximately 15% for the hyperemic flow. Thus, current results with the moderate stenosed artery indicate that reducing Hct might be favorable in terms of increasing O(2) flux and pO(2,min), in the medial region of the wall for the basal flow, while higher Hct is advantageous for the hyperemic flow beyond the diverging section. The results of this study not only provide significant details of oxygen transport under varying pathophysiologic blood conditions such as unusually high blood viscosity and flow rate, but might also be extended to offer implications for drug therapy related to blood-thinning medication and for blood transfusion and hemorrhage.

Longitudinal Assessment of Hemodynamic Endpoints in Predicting Arteriovenous Fistula Maturation

Arteriovenous fistula (AVF) nonmaturation is currently a significant clinical problem; however, the mechanisms responsible for this have remained unanswered. Previous work by our group and others has suggested that anatomical configuration and the corresponding hemodynamic endpoints could have an important role in AVF remodeling. Thus, our goal was to assess the longitudinal (temporal) effect of wall shear stress (WSS) on remodeling process of AVFs with two different configurations. The hypothesis is that early assessment of hemodynamic endpoints such as temporal gradient of WSS will predict the maturation status of AVF at later time points. Two AVFs with curved (C‐AVF) and straight (S‐AVF) configurations were created between the femoral artery and vein of each pig. Three pigs were considered in this study and in total six AVFs (three C‐AVF and three S‐AVF) were created. The CT scan and ultrasound were utilized to numerically evaluate local WSS at 20 cross‐sections along the venous segment of AVFs at 2D (D: days), 7D, and 28D postsurgery. These cross‐sections were located at 1.5 mm increments from the anastomosis junction. Local WSS values at these cross‐sections were correlated with their corresponding luminal area over time. The WSS in C‐AVF decreased from 22.3 ± 4.8 dyn/cm2 at 2D to 4.1 ± 5.1 dyn/cm2 at 28D, while WSS increased in S‐AVF from 13.0 ± 5.0 dyn/cm2 at 2D to 36.7 ± 5.3 dyn/cm2 at 28D. Corresponding to these changes in WSS levels, luminal area of C‐AVF dilated (0.23 ± 0.14 cm2 at 2D to 0.87 ± 0.14 cm2 at 28D) with attendant increase in flow rate. However, S‐AVF had minimal changes in area (0.26 ± 0.02 cm2 at 2D to 0.27 ± 0.03 cm2 at 28D) despite some increase in flow rate. Our results suggest that the temporal changes of WSS could have significant effects on AVF maturation. Reduction in WSS over time (regardless of initial values) may result in dilation (p < 0.05), while increase in WSS may be detrimental to maturation. Thus, creation of AVFs in a specific configuration which results in a decline in WSS over time may reduce AVF maturation failure.

Measurement of hemodynamic and anatomic parameters in a swine arteriovenous fistula model.

Purpose Although arteriovenous fistulae (AVFs) are currently the preferred mode of permanent hemodialysis access they do have significant problems due to initial non-maturation and a later venous stenosis. These problems appear to have been exacerbated following a push to increase AVF prevalence in the US. The reasons for both AVF non-maturation and the later venous stenoses are unclear but are thought to be related to abnormal hemodynamic wall shear stress (WSS) profiles. This technical note aims to describe the successful development of measurement techniques that can be used to establish a complete hemodynamic profile in a pig model with two different configurations of AVF. Methods and results The curved and straight AVF configurations were created in an in vivo pig model. Flow and pressure in the AVFs were measured using the perivascular flow probes and Doppler flow wires while the pressure was recorded using a pressure transducer. The anatomical configuration was obtained using two different approaches: a) combination of intravascular ultrasound (IVUS) and angiograms, (b) 64 slice CT angiography. 3D models were reconstructed using image processing and computer modeling techniques. Numerical calculations were then performed by applying the measured flow and pressure data into the configurations to obtain the hemodynamic WSS profiles. Conclusion The described methodologies will allow the calculation and optimization of WSS profiles in animal models. This information could then be translated to the clinical setting where it would have a positive impact on improving the early maturation rates of AVFs as well as reducing the late venous stenoses.

Photoinstability of S-Nitrosothiols during Sampling of Whole Blood: A Likely Source of Error and Variability in S-Nitrosothiol Measurements

BACKGROUND The determination of reference intervals for the concentration of total S-nitrosothiols (RSNOs) in blood is a highly controversial topic, likely because of the inherent instability of these species. Most currently available techniques to quantify RSNOs in blood require considerable sample handling and multiple pretreatment steps during which light exposure is difficult to completely eliminate. We investigated the effect of brief light exposure on the stability of RSNO species in blood during the initial sampling process. METHODS A novel amperometric RSNO sensor, based on an immobilized organoselenium catalyst at the distal tip of an electrochemical nitric oxide detector, was used to determine RSNO species in diluted whole blood without centrifugation or pretreatment. Porcine blood was collected into aluminum foil-wrapped syringes via a 12-inch butterfly needle tube assembly. Two blood samples were collected from the same animal -- one with the butterfly needle tubing wrapped in aluminum foil and one with the tubing exposed to ambient room light. The RSNO concentrations in these sequential blood samples were determined by a standard addition procedure. RESULTS Eight sets of measurements were made in 6 animals. Samples exposed to light yielded RSNO concentrations only 23.6% (7.2%) [mean (SD)] of the RSNO concentrations determined in samples that were shielded from light and obtained from the same animals. CONCLUSIONS These results suggest significant photoinstablity of RSNOs in whole blood and indicate the critical importance of proper light protection during sampling and processing of blood samples for the accurate determinations of endogenous RSNO concentrations.

Neointimal Hyperplasia and Calcification in Medium Sized Arteries in Adult Patients with Chronic Kidney Disease

The nature of arterial changes resulting in cardiovascular events and dialysis vascular access failures in adult predialysis patients is not well known. This study examined intimal changes, calcium deposition, and consequent stiffness in brachial and radial arteries of adult CKD patients. Ten brachial‐artery and seven radial‐artery specimens were obtained during fistula creation from nine predialysis and eight dialysis‐dependent, nondiabetic patients; and age‐gender matched controls undergoing coronary bypass grafts (6 radial) or kidney donation (6 renal). Arterial stiffness was measured at baseline. Vessel histology, morphometric analysis of intima‐media, and direct quantification of calcium load was performed using standard techniques. Both predialysis and dialysis patients demonstrated significant arterial intimal hyperplasia with intima:media ratio higher than controls (0.13 ± 0.12 vs. 0.02 ± 0.05, p = 0.01). Calcium deposition was demonstrated on histology and the calcium content in patients was higher than controls (34.68 ± 26.86 vs. 10.95 ± 9.18 μg/μg, p = 0.003). The blood vessel calcium content correlated with arterial stiffness (r = 0.64, p = 0.018). This study for the first time describes, and suggests mechanistic linkage between, intimal hyperplasia, pathological calcium deposition, and increased functional arterial stiffness in dialysis and predialysis patients. Our research could serve as a unique window into the in vivo status of the uremic vasculature impacting fistula maturation and cardiovascular disease.