Ritman, Erik L.

Abstract A modified myocardium-blood composite model enables the temporal changes in instantaneous extension and contraction of in vivo myocardial fiber to be computed by using the measured blood volume fractions and wall thicknesses in the investigated left ventricular region. A 16 time-increment study of 6 dogs shows that myocardial fiber may undergo an average length change of 62% and −38% during the diastolic and systolic phases of cardiac cycle, respectively. Two comparable results had been obtained previously, but the new method is much simpler, removes the incompressibility assumption and takes into consideration the intravascular blood in the ventricular wall.

Abstract For biomechanics analysis of body organs, finite element method [1∼3] is the practical tool for dealing with the complex geometric shapes and material composition involved. Either for direct computation of the stresses and strains in the body organ or inverse determination of its material properties, the change of geometrical shapes and dimensions and the change of the loadings, both in time, must be accurately recorded. Moreover, fast imaging techniques [4] and a vast volume of computer storage memory are necessary for acquisition of such temporal data, particularly if finite-element biomedical algorithms are to be developed for routine use. This paper reports a chronological development of finite-element inverse determination of global and regional material properties, passive diastolic stiffness and active systolic contractile strength of the in vivo heart wall. A collection of computer algorithms has been accumulated as a result of continuing collaborative research on cardiopulmonary dynamics by the authors and their co-workers since 1973.

Abstract For occlusion experiments of 9 dogs [1], the infarcted and not-infarcted regions of left ventricular cross sections have been investigated by application of a plane-strain finite-element method to determine the change in the wall passive stiffness, or, Young’s modulus E [2]. The present paper reports how the instantaneous strain equation derived from the myocardium-blood composite model [3,4] can be utilized to obtain a formula for calculation of the ratio of extended myocardial fiber length in the infarcted (IN) region and that in the not-infarcted (NI) region of left ventricular cross section during the diastole phase of cardiac cycle. This formula uses the blood volume fraction (f) data [5] listed in Table 1 and the ventricular wall thickness (t) directly measurable from the LV walls as visualized in short-axis tomographic images of the LV.

Site specific targeting remains an elusive goal for stem cell and gene therapies in the cardiovascular field. One promising option involves use of modified 3D stent designs that deliver larger cell/gene payloads to specific disease sites using the versatility of percutaneous vascular access. Here, we engineered smooth muscle cells (SMCs) placed in a 3D metallic stent scaffold to deliver high local concentrations of an angiogenic gene (VEGF165) in a porcine model of chronic total occlusion involving ameroid placement on the proximal left circumflex (LCx) artery(n≥8 per group). Engineered SMCs were retained within the mesh stent structure (479 ± 47.4 SMC/mm2) and were competent for VEGF production in vitro (8.8 ± 1.8 ng/ml in media). Following implantation, micro CT analyses revealed that stents loaded with VEGF producing SMCs significantly enhanced vasa vasora microvessel density (5.43 ± 0.46 vs 2.14 ± 0.19 vessels/mm2, p<0.001) with a concomitant increase in tissue VEGF protein levels (277.9 ± 85.7 vs 18.8 ± 4.4 pg/mg protein, p<0.0001). Formation of endothelial cell colonies from peripheral blood mononuclear cells was significantly increased with the implantation of VEGF-secreting cells suggesting increased angiogenic potential (7.0 ± 2.0 versus 41.6 ± 4.5, EC:CFU p<0.0001). VEGF secreting mesh stent placement enhanced regional blood flow analyses determined by microsphere analyses as well as improving regional wall motion in the LCx-related territory (p<0.05) as well as left ventricular function assessed in terms of ejection fraction (34.2 ± 6.7% vs 61.2 ± 5.2% P<0.001) and dp/dt (p<0.001) Our data indicate robust clinically relevant angiogenesis can be achieved in a human scale porcine chronic vascular occlusion model following stent based delivery of angiogenic cells. This may have implications for percutaneous delivery of stem cells, genes and secreted therapeutic factors in cardiovascular disease in general and in creation of percutaneous microvascular bypass networks in the setting of chronic vaso-occlusive disease in particular.

Patients with congenital heart disease who have a morphological right ventricle (RV) serving as a systemic ventricle have an increased incidence of RV dysfunction. A different structural response of micro‐vessels to increased stress in the RV wall is a possible mechanism for this dysfunction. We examined this possibility by measuring the volume of myocardium perfused by arteries less than 200μm in lumen diameter (V) 7 weeks after a 5mg/kg IV injection of monocrotaline in three 2‐week old and three 18‐week old pigs. This information was obtained by use of 3D micro‐CT of transmural segments (~ 2cm 3 ) of the RV wall. The table provides the average values of V (mm 3 /mm 2 ± SD). Pulmonary artery pressures in the mature, treated pigs were 35±6 mmHg and in the juvenile were 25±2 mmHg. V did not differ from control in the treated 2.5‐month old pigs but trended down in the 6‐month old pigs. We conclude that the volume of myocardium perfused by an arteriole in the RV wall may be an indicator of the perfusion reserve available to the microcirculation in response to increased wall stress. A more definitive answer would require delayed study of the early onset treatment group at the age of 6 months. (Supported in part by NIH grant EB000305)

Obesity is a risk factor for cardiovascular disease, hypertension and diabetes. Although a variety vasculogenic factors have been associated with adipose tissue, there is little information available concerning the 3‐dimensional vasculature architecture of adipose tissue. Ten Swiss mice were fed a high fat diet (11% fat content) and ten mice were fed a normal chow diet (4% fat content). After 40 weeks on the diet, the high fat mice (46.7 ± 3.8 gm; mean ± SEM) were significantly larger than the mice on the normal diet (34.5 ± 0.8 gm; mean ± SEM). The mice were anesthetized and the para‐uterine fat pads were infused via the abdominal aorta first with heparinized 0.9% saline, then with 10% buffered formalin and finally with radiopaque silicone rubber (Microfil 122). One para‐uterine fat pad from each mouse was prepared for histological evaluation and the remaining fat pad was prepared for 3‐dimensional micro‐computed tomography. The fat pads were significantly larger in the high fat mice (7.6 ± 1.2 gm; mean ± SEM) than in the normal mice (2.6 ± 0.3 gm; mean ±SEM). There was no difference between the two groups in vascular number density (number of vessels per μl) of the fat pads. However, because of the difference in tissue mass, there was a significant difference between groups in total vessel number. These results indicate that the vascular growth proportionally accompanies tissue growth in adipose tissue. Supported in part by a Faculty Research Grant, Minnesota State University, Mankato, and in part by NIH grant EB 000305.

Despite the importance of the vasculature during tumorigenesis and during antiangiogenesis treatment, there are few methods available to visualize conveniently the three‐dimensional microvascular structure of tumors. Micro‐computed tomography provides a means to visualize conveniently and quantitatively the three‐dimensional tumor microvascular architecture during tumorigenesis and treatment. In the present study, micro‐computed tomography was utilized to visualize the vasculature of hepatic tumors in SV‐40 transgenic rats. The rats were anesthetized and the vasculature of the livers was infused via an aortic cannula, first with heparinized 0.9% saline, then with 10% buffered formalin and finally with radiopaque silicone rubber (Microfil 122). The livers were first scanned by micro‐CT to visualize the microvasculature and then prepared for histology to visualize the parenchyma. At 10 months of age, all transgenic rats developed gross tumors in their livers. Histological examination revealed that the malignant hepatic lesions in the livers displayed mixed structures resembling hepatoblastoma, cholangiocellular or hepatocellular differentiation in human. Various sized vessels were visible extending to the tumors. Three‐dimensional micro‐computed tomography showed that those vessels were disorganized and tortuous with atypical branching patterns. Often vascular “lakes” were found in the mass of the large tumors. These results show the utility of micro‐computed tomography for studying tumor vasculogenesis. Supported in part by NIH grant EB 000305.

Background Kidney stones (nephrolithiasis) are an expensive and painful health problem with a complex etiology with ~70% of stones composed of calcium oxalate (CaOx). The Slc26 gene family contains several proteins that transport oxalate in the gut and kidney. Slc26a6 and its homologs exchange Cl − for oxalate (ox 2− ), as well as SO 4 2− , HCO 3 − and formate. Slc26a6 −/− mice have hyperoxaluria and CaOx stones illustrating a physiologic collaboration between the gut and kidney. We developed a Drosophila genetic model for CaOx stones (dPrestin, Slc26a5/6, manipulation) allowing rapid gut, renal and whole‐body physiological assessment (min/h v. mo/y). Methods (Tissue) First, Malpighian tubules (MT, renal tubule) were dissected from wild type and dPrestin knockdown mutants and soaked in ox 2− solutions. CaOx stones were visualized by DIC microscopy. Second, we generated Drosophila expressing eYFP in the MT, MT+gut or whole body. eYFP is a halide (Cl − ) sensor, allowing quantification of Cl − /ox 2− exchange by native dPrestin. (Whole body) Wild type and dPrestin knockdown flies were fed ox 2− enriched food, and flies imaged (CaOx quantified) by micro computed tomography (MicroCT). Results Exposing dPrestin‐knockdowns to bath ox 2− elicited almost no CaOx crystals. However, exposing wt‐MT to ox 2− elicited many CaOx crystals in 20 min. Increased CaOx crystal formation was also coupled with an eYFP intensity increase, matching dPrestin Cl − /ox 2− exchange characteristics. MicroCT showed more CaOx crystals in flies that fed on ox 2− longer. Conclusions Our observations indicate that Drosophila and dPrestin manipulation can be used for rapid, detailed studies of gut, renal and whole organism oxalate metabolism & CaOx stone formation. [DK83007]

Background Kidney stones are an expensive and painful health problem with calcium oxalate (CaOx) being 70% of cases. We developed a Drosophila model of CaOx stones [Hirata et al., AJP‐Renal Physiol, 2012]. This work showed that dPrestin (Slc26a5/a6) exchanges Cl − for oxalate (Ox). Thiosulfate (S 2 O 3 ) and tannic acid (TA) are implicated to inhibit CaOx crystals in supersaturated urine. Here, we report transporter, renal tubule and whole body effects of S 2 O 3 and TA on crystal formation. Methods S 2 O 3 and TA transport effects were examined by voltage clamping of dPrestin‐expressing Xenopus oocytes. Cis ‐inhibition of oxalate transport was also examined in (a) dissected Malpighian tubules (MT) or flies fed ox‐enriched food plus S 2 O 3 or TA. MT CaOx crystals were viewed by DIC, or whole flies by microCT. Results dPrestin transports S 2 O 3 but not TA, yet both inhibited Ox transport (K i (S 2 O 3 )~10mM; K i (TA)~100μM). S 2 O 3 competitively inhibits while TA blocks dPrestin. MT treated with S 2 O 3 or TA ↓ crystal formation compared to MT soaked in Ox only. Flies fed with S 2 O 3 or TA also showed ↓ CaOx in MTs. Conclusions S 2 O 3 and TA are both inhibitors of CaOx crystallization. These experiments show that these inhibitors have distinct mechanisms of action. Our Results indicate that (a) this Drosophila model can be used as a chemical screening assay for CaOx stone prevention and (b) TA, due to low Ki, might be a reasonable therapy for certain CaOx stones patients.[ DK83007, DK92408]