Lorenz Gürke

Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine.

Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF) is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localization of angiogenic signals in the extracellular matrix (ECM) is crucial to ensure the proper assembly and maturation of new vascular structures. Here, we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of ECM to optimize the signaling microenvironment of vascular growth factors.

Risk stratification by the virtual crossmatch: a prospective study in 233 renal transplantations

The virtual crossmatch (virtual‐XM) has been proposed for accurate identification of donor‐specific HLA‐antibodies, but large prospective studies assessing its value for pretransplant risk stratification are lacking. A total of 233 consecutive renal allograft recipients were prospectively stratified according to the virtual‐XM. In patients with a negative virtual‐XM (n = 190, 82%), prospective cytotoxicity crossmatches (CDC‐XM) were omitted, and they received standard immunosuppression. Virtual‐XM positive patients were only transplanted if CDC‐XM were negative. They received additional induction with anti‐T‐lymphocyte‐globulin and intravenous immunoglobulins (n = 43, 18%). The cumulative incidence of clinical/subclinical antibody‐mediated rejection (AMR) at 1 year was lower in the negative virtual‐XM than in the positive virtual‐XM group [15/190 (8%) vs. 18/43 (42%); P < 0.0001]. After a median follow‐up of 2.6 years, allograft loss because of AMR occurred less often in the negative virtual‐XM group (1% vs. 7%; P = 0.04) and death‐censored allograft survival at 2 years was higher (98% vs. 91%; P = 0.02). Serum creatinine was not different at the last follow‐up (129 μm vs. 130 μm; P = 0.58). We conclude that a negative virtual‐XM defines patients at low risk for AMR and early allograft loss, while a positive virtual‐XM represents a significant risk for AMR despite enhanced induction therapy. This supports the utility of the virtual‐XM for risk stratification and treatment allocation.

A preliminary report on the prognostic significance of preoperative brain natriuretic peptide and postoperative cardiac troponin in patients undergoing major vascular surgery.

BACKGROUND: Associations between preoperative elevation of brain natriuretic peptide (BNP) or postoperative elevation of cardiac troponins (cTn) with major adverse cardiac events (MACE) after major surgery have been shown previously. In this study, we evaluated the added value of preoperative BNP with postoperative cTn levels for the prediction of MACE in patients undergoing major vascular surgery. METHODS: This is a prospectively prespecified, secondary analysis of data from a cohort of 133 clinically stable patients undergoing major vascular surgery enrolled in a clinical trial evaluating the effectiveness of the sympathetic nervous system-inhibiting drug moxonidine on reducing MACE. Concentrations of BNP and cTn were determined before surgery, and concentrations of cTn were measured immediately after surgery and on postoperative days 1, 2, 3, and 7. The primary end point was the occurrence of MACE (defined as any hospitalization for myocardial revascularization, acute coronary syndrome, acute congestive heart failure, or death by any cause) within 1 yr after surgery. Patients were evaluated for MACE by hospital chart review during hospitalization and by telephone interviews 12 mo after surgery. RESULTS: Within 1 yr after surgery, 19 patients (14%) had a MACE, including 14 patients (11%) who died. After adjustment for age, gender, and the revised cardiac risk index, preoperative BNP elevation ≥50 pg/mL was associated with MACE (adjusted hazard ratio [HR]: 6.5, 95% confidence interval [CI]: 1.4–29.5) regardless of the subsequent cTn I concentrations. The combination of preoperative BNP elevation ≥50 pg/mL and postoperative cTn I elevation ≥2 ng/mL was associated with MACE (adjusted HR: 25.2, 95% CI: 5.0–128.4) and all-cause mortality (adjusted HR: 18.7, 95% CI: 3.1–112.5). The negative predictive value of a normal preoperative BNP value for subsequent adverse events was 0.965 (95% CI: 0.879–0.996). CONCLUSION: These data suggest that measurement of preoperative BNP concentrations in addition to postoperative cTn concentrations provides additive prognostic information for MACE and mortality after major vascular surgery.

Major vascular resection and prosthetic replacement for retroperitoneal tumors.

IntroductionInvolvement of major vascular structures has been considered a limiting factor for resecting advanced tumors. The objective of this study was to evaluate the outcome after concomitant retroperitoneal tumor and vascular resection with prosthetic replacement of the aorta/vena cava.MethodsThe authors reviewed a 5-year series of eight patients with a median age of 50 years (range 11–68 years) who had undergone resection of a retroperitoneal tumor and concomitant resection and replacement of the abdominal aorta, inferior vena cava, or both. The histologic diagnoses were sarcoma (five patients), teratoma (one), transitional cell carcinoma (one), and ganglioneuroma (one). The main outcome measures were early (< 30 days) and late (≥ 30 days) surgical morbidity and mortality. Secondary endpoints were vascular graft patency and tumor-free survival. Two patients underwent combined graft replacement of the aorta and vena cava. Single aortic and vena cava graft replacement were each done in three patients.ResultsTwo patients showed early surgical morbidity necessitating reoperation for a thrombotic graft occlusion. No patient died during the early course of the follow-up. During a median follow-up of 14 months (range 1–56 months), two patients had late surgical morbidity. The median tumor-free survival for patients with malignancy was 14 months (range 1–54 months). One patient developed locoregional tumor recurrence, and two developed distant metastases. The median survival for patients with malignancy was 14 months (range 1–60 months).ConclusionsAn aggressive surgical approach for otherwise unresectable retroperitoneal tumors with vascular resection and prosthetic vascular replacement is justified in selected cases and has acceptable morbidity and mortality.

High-throughput flow cytometry purification of transduced progenitors expressing defined levels of vascular endothelial growth factor induces controlled angiogenesis in vivo

Delivery of therapeutic genes by genetically modified progenitors is a powerful tool for regenerative medicine. However, many proteins remain localized within or around the expressing cell, and heterogeneous expression levels can lead to reduced efficacy or increased toxicity. For example, the matrix‐binding vascular endothelial growth factor (VEGF) can induce normal, stable, and functional angiogenesis or aberrant angioma growth depending on its level of expression in the microenvironment around each producing cell, and not on its total dose. To overcome this limitation, we developed a flow cytometry–based method to rapidly purify transduced cells expressing desired levels of a therapeutic transgene. Primary mouse myoblasts were transduced with a bicistronic retrovirus expressing VEGF linked to a nonfunctional, truncated form of the syngenic molecule CD8a. By using a clonal population uniformly expressing a known VEGF level as a reference, cells producing similar VEGF amounts were rapidly sorted from the primary population on the basis of their CD8a fluorescence intensity. A single round of sorting with a suitably designed gate yielded a purified population that induced robust, normal, and stable angiogenesis, and completely avoided angioma growth, which was instead always caused by the heterogeneous parent population. This clinically applicable high‐throughput technique allowed the delivery of highly controlled VEGF levels in vivo, leading to significantly improved safety without compromising efficacy. Furthermore, when applied to other suitable progenitor populations, this technique could help overcome a significant obstacle in the development of safe and efficacious vascularization strategies in the fields of regenerative medicine and tissue engineering. STEM CELLS 2010;28:611–619

Why do kidney grafts fail? A long-term single-center experience

Abstract Chronic allograft failure remains the main problem limiting long‐term success after kidney transplantation. The aim of this retrospective analysis was to define clinical and histological parameters associated with favorable or poor 10‐year outcome. To compare outcome we defined two groups of cadaveric‐allograft recipients: a good‐outcome group (GOG), composed of 145 cadaveric‐kidney recipients who had lived with a functioning graft for at least 10 years and who were either still alive or had died with the functioning graft, and a poor‐outcome group (POG) consisting of 86 cadaveric‐kidney recipients who had had a functioning graft for at least 1 year and had returned to dialysis between 1 and 10 years after transplantation. The following factors were found to be statistically significant indicators of poor outcome: advanced donor age (P= 0.0001); a first biopsy‐proven acute rejection episode within the 1st year (P<0.0001); more than one acute rejection episode within the 1st year (P<0.0001); acute vascular rejection, especially if occurring after the 3rd month (P<0.0001); chronic sclerosing rejection (P<0.0001); glomerulonephritis in the graft (P= 0.0001); and non‐compliance and suboptimal medical treatment (15% of the POG). The mean plasma creatinine and mean urine protein‐to‐creatinine ratios were significantly lower at 1 months and 1 year in the GOG. In conclusion, advanced donor age, acute rejection episodes with vascular involvement, chronic sclerosing rejection, non‐compliance, and suboptimal medical treatment are strong predictors of a poor long‐term outcome. The plasma creatinine and protein‐to‐creatinine ratios at 1 year are the best predictors of good or poor long‐term outcome.

How Painful Is Donor Nephrectomy? Retrospective Analysis of Early Pain and Pain Management in Open versus Laparoscopic versus Retroperitoneoscopic Nephrectomy

Background. The aim of this study was to evaluate the early postoperative pain and pain management after standard open (ODN), hand-assisted laparoscopic (HLDN) and retroperitoneoscopic (RDN) donor nephrectomy. Methods. The visual analogue scale (VAS) was determined twice a day in 203 donors during the first five days after nephrectomy. Results. Mean VAS was significantly lower after RDN and HLDN than after ODN on day 2 (p=0.004) and days 3–5 (p<0.001). After RDN, “no pain” (VAS=0) was reported significantly earlier than after ODN. Irrespective of the technique used and the pain management, all donors reported significantly higher VAS in the morning. Opiates were administered for a significantly shorter average time period after RDN than after ODN (p=0.005). Cumulative morphine equivalent doses were higher after ODN than after RDN (p=0.001). Mean VAS reported after HLDN and RDN was similar. Conclusions. In summary, RDN and HLDN were clearly associated with much less early pain than ODN, independently of the used pain management.

Preconditioning with Short Cycles Improves Ischemic Tolerance in Rat Fast- and Slow-Twitch Skeletal Muscle

Aim: The aim of this study was to investigate whether the efficacy of ischemic preconditioning (IP) in rat skeletal muscle depends on the duration of the preconditioning cycles. Methods: Rats were divided into four groups (n = 10 each). The right hindlimb of rats in group A were subjected to 2.5 h of tourniquet ischemia followed by 2 h of reperfusion (I-R). Thereafter, muscular function was analyzed in vitro and high-energy phosphates (HEP) were determined by HPLC. Before I-R, right hindlimbs of rats in groups B–D subjected to IP with three cycles each consisting of 2.5, 5 or 10 min of ischemia followed by reperfusion for the same duration. Results: Postischemic function of the extensor muscle was significantly improved with all three preconditioning protocols. Postischemic function of the soleus muscle was only improved by IP with three cycles of 5 min of ischemia and 5 min of reperfusion. Postischemic HEP tissue levels were not influenced by IP. Conclusion: This study shows for the first time that IP increases ischemic tolerance not only of fast-twitch but also of slow-twitch skeletal muscle. The efficacy of IP seems to be less dependent on the duration of the single preconditioning cycle than on the number of cycles performed. Three cycles each of 2.5, 5 or 10 min ischemia and reperfusion significantly improved postischemic skeletal muscle function. Tissue levels of HEPs, however, were not influenced by IP indicating that preservation of HEPs does not play a major role in the effects of IP on rodent skeletal muscle.

Retroperitoneoscopic living-donor nephrectomy: first clinical experiences in 19 operations.

The wish for a minimally invasive procedure is one of the utmost demands by all persons undergoing living-donor nephrectomies. The retroperitoneoscopic access for this procedure has proven to be a safe, minimally invasive, and efficient approach accompanied by early mobilization and a fast return to general daily activities. The incidence of complications during and after this technically demanding operation is comparable to that of other approaches. Because of the direct anatomic approach, retroperitoneoscopic living-donor nephrectomy has become the preferred access at our institution for both donors and surgeons.

Function of Fast- and Slow-Twitch Rat Skeletal Muscle following Ischemia and Reperfusion at Different Intramuscular Temperatures

Fast- (peroneal) and slow-twitch (soleus) skeletal muscles of anesthetized Wistar rats were subjected to 3 h of tourniquet ischemia. The intramuscular temperature of the leg was adjusted to 22, 30 or 35°C (n = 12 per group) during ischemia. After 2 h of reperfusion, the muscles were electrically stimulated in vitro and muscular function was analyzed for maximal force, performance, contractility and fatigue. Contralateral nonischemic muscles served as controls. Three hours of ischemia at 30°C did not reduce the function of the peroneal muscles compared to nonischemic controls. The same ischemic stress significantly reduced the function of the soleus muscles compared to nonischemic controls. The postischemic function of the soleus muscles declined with increasing temperature. The postischemic function of the 35°C group of peroneal muscles was significantly reduced compared to the 22 and the 30°C groups, which did not differ. These results provide evidence that fast-twitch muscles are more resistant to ischemia than slow-twitch muscles. They furthermore show a fiber type-specific dependency of postischemic muscle function on intramuscular temperature during ischemia. Hypothermia-sensitive fast-twitch fibers predominate in the skeletal muscles of the extremities. Mild hypothermia could, therefore, reduce tourniquet ischemia-induced injury after surgery of the extremities.