Homocysteine is associated with severity of microvasculopathy in sickle cell disease patients.

The pathophysiology of sickle cell disease (SCD) includes vasculopathy as well as anaemia. Elevated plasma homocysteine is a risk factor for vascular disease and may be associated with increased risk of vascular complications in SCD patients. In the present study, microvascular characteristics were assessed in the bulbar conjunctiva of 18 paediatric and 18 adult SCD patients, using the non-invasive technique of computer-assisted intravital microscopy. A vasculopathy severity index (SI) was computed to quantify the degree of microvasculopathy in each patient. Plasma homocysteine and several of its determinants [serum folate and vitamin B12, plasma pyridoxal-5'-phosphate (vitamin B6 status) and creatinine (kidney function)] were measured. Age was strongly correlated with microvasculopathy in the SCD patients, with the SI increasing about 0·1 unit per one-year increase in age (P < 0·001). After adjusting for age, gender, B-vitamin status and creatinine, homocysteine concentration was directly correlated with severity index (P < 0·05). Age and homocysteine concentration were independent predictors of microvasculopathy in SCD patients. It remains to be determined whether lowering homocysteine concentrations using appropriate B-vitamin supplements (folate and vitamins B12 and B6) - particularly if started early in life - could ameliorate microvasculopathy and its associated complications in SCD patients.

Insulin-producing intestinal K cells protect nonobese diabetic mice from autoimmune diabetes.

BACKGROUND & AIMS: Type 1 diabetes is caused by an aberrant response against pancreatic ß cells. Intestinal K cells are glucose-responsive endocrine cells that might be engineered to secrete insulin. We generated diabetes-prone non-obese diabetic (NOD) mice that express insulin, via a transgene, in K cells. We assessed the effects on immunogenicity and diabetes development. METHODS: Diabetes incidence and glucose homeostasis were assessed in NOD mice that expressed mouse preproinsulin II from a transgene in K cells and nontransgenic NOD mice (controls); pancreas and duodenum tissues were collected and analyzed by histology. We evaluated T cell responses to insulin, levels of circulating autoantibodies against insulin, and the percentage of circulating antigen-specific T cells. Inflammation of mesenteric and pancreatic lymph node cells was also evaluated. RESULTS: The transgenic mice tended to have lower blood levels of glucose than control mice, associated with increased plasma levels of immunoreactive insulin and proinsulin. Fewer transgenic mice developed diabetes than controls. In analyses of pancreas and intestine tissues from the transgenic mice, insulin-producing K cells were not affected by the immune response and the mice had reduced destruction of endogenous ß cells. Fewer transgenic mice were positive for insulin autoantibodies compared with controls. Cells isolated from mesenteric lymph nodes of the transgenic mice had significantly lower rates of proliferation and T cells from transgenic mice tended to secrete lower levels of inflammatory cytokines than from controls. At 15 weeks, transgenic mice had fewer peripheral CD8(+) T cells specific for NRP-V7 than control mice. CONCLUSIONS: NOD mice with intestinal K cells engineered to express insulin have reduced blood levels of glucose, are less likely to develop diabetes, and have reduced immunity against pancreatic ß cells compared with control NOD mice. This approach might be developed to treat patients with type 1 diabetes.

An integrin binding-defective mutant of insulin-like growth factor-1 (R36E/R37E IGF1) acts as a dominant-negative antagonist of the IGF1 receptor (IGF1R) and suppresses tumorigenesis but still binds to IGF1R.

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (αvß3 and α6ß4) and induces ternary complex formation (integrin-IGF1-IGF1 receptor (IGF1R)) and that the integrin binding-defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with WT IGF1 for binding to IGF1R and inhibits IGF signaling. Here, we described that excess R36E/R37E suppressed cell viability increased by WT IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect an effect of WT IGF1 or R36E/R37E in cancer cells under anchorage-dependent conditions. However, under anchorage-independent conditions, WT IGF1 enhanced cell viability and induced signals, whereas R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by WT IGF1 under anchorage-independent conditions. Using cancer cells stably expressing WT IGF1 or R36E/R37E, we determined that R36E/R37E suppressed tumorigenesis in vivo, whereas WT IGF1 markedly enhanced it. R36E/R37E suppressed the binding of WT IGF1 to the cell surface and the subsequent ternary complex formation induced by WT IGF1. R36E/R37E suppressed activation of IGF1R by insulin. WT IGF1, but not R36E/R37E, induced ternary complex formation with the IGF1R/insulin receptor hybrid. These findings suggest that 1) IGF1 induces signals under anchorage-independent conditions and that 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.

A potent oral P-selectin blocking agent improves microcirculatory blood flow and a marker of endothelial cell injury in patients with sickle cell disease.

Abnormal blood flow accounts for most of the clinical morbidity of sickle cell disease (SCD) [1,2]. Most notably, occlusion of flow in the microvasculature causes the acute pain crises [3] that are the commonest cause for patients with SCD to seek medical attention [4] and major determinants of their quality of life [5]. Based on evidence that endothelial P-selectin is central to the abnormal blood flow in SCD we provide results from four of our studies that are germane to microvascular blood flow in SCD. A proof-of-principle study established that doses of heparin lower than what are used for anticoagulation but sufficient to block P-selectin improved microvascular blood flow inpatients with SCD. An in vitro study showed that Pentosan Polysulfate Sodium (PPS) had greater P-selectin blocking activity than heparin. A Phase I clinical study demonstrated that a single oral dose of PPS increased microvascular blood flow in patients with SCD. A Phase II clinical study that was not completed documented that daily oral doses of PPS administered for 8 weeks lowered plasma levels of sVCAM-1 and tended to improve microvascular blood flow in patients with SCD. These data support the concept that P-selectin on the microvascular endothelium is critical to both acute vascular occlusion and chronically impaired microvascular blood flow in SCD. They also demonstrate that oral PPS is beneficial to microvascular sickle cell blood flow and has potential as an efficacious agent for long-term prophylactic therapy of SCD.

Exchange transfusion therapy and its effects on real-time microcirculation in pediatric sickle cell anemia patients: an intravital microscopy study.

Periodic blood exchange transfusion is a treatment modality commonly used to manage pediatric sickle cell anemia at the University of California Davis Medical Center. The goal of exchange transfusion therapy is to ameliorate vasoocclusion and improve tissue perfusion by removing sickled red blood cells and introducing normal red blood cells. Using computer-assisted intravital microscopy, pretransfusion and posttransfusion microvascular characteristics were analyzed. In this study, the bulbar conjunctiva exhibited a "blanched" avascular appearance in all 6 pediatric sickle cell anemia patients before transfusion, indicative of tissue hypoperfusion and ischemia. Immediately after transfusion, substantial improvement in vascularization and tissue perfusion resulted, reflected by the enhanced appearance of capillaries and arterioles. In addition, a decrease in red cell velocity was observed. These observations provide evidence that exchange transfusion therapy is beneficial in ameliorating vasoocclusion and improving tissue perfusion. However, with the paradoxical posttransfusion decrease in red cell velocity presumably due to induced hyperviscosity from the large transfusion volume, blood flow is still impaired. This decreased velocity may thwart efforts to improve oxygen delivery through transfusion and may, to some extent, promote vasoocclusion instead. This paradoxical result warrants further investigation on the effects of transfusion volume and viscosity in the exchange transfusion process.

Correlation of conjunctival microangiopathy with retinopathy in type-2 diabetes mellitus (T2DM) patients.

We hypothesized that T2DM vasculopathy can be revealed and quantified in the bulbar conjunctiva prior to its pathologic presentation in the retina. Using computer-assisted intravital microscopy (CAIM), an objective, non-invasive approach can provide a viable complement to retinal fundus photography to possibly screen patients for early signs of real-time, in vivo T2DM vasculopathy. Fundus photography was utilized to determine the retinopathy level (RL) in T2DM patients with non-proliferative diabetic retinopathy (NPDR) and control subjects. CAIM was used to quantify microangiopathy in the bulbar conjunctiva in the same patients, and reported on a severity index (SI). The average RL for the T2DM patients in this study is 19.68 ± 9.91, which differs from control subjects (RL = 10 ± 0.0; p < 0.05). A significant difference in vasculopathy was observed in the conjunctival microcirculation in the same patients (SI = 5.81 ± 1.30) when compared with control subjects (SI = 1.33 ± 1.58; p < 0.05). The results provide evidence that significant vasculopathy had developed in the microcirculation in the bulbar conjunctiva, though diabetic retinopathy had not developed significantly in the same patients - indicative of the presence of a time window for early intervention of T2DM before non-proliferative retinopathy develops, and the real-time availability of the conjunctival microvasculature as an in vivo platform to monitor disease progression.

Alzheimer's disease: more than amyloid.

CONTEXT: The etiology of Alzheimer's disease (AD) is inconclusive. Treatments targeting amyloid have largely been unsuccessful. There is increasing evidence that vasculopathy may play an important pathogenic role in AD. OBJECTIVE: Longitudinal measurements of whole blood viscosity (WBV) using a computer-assisted hemorheologic protocol and characterization of microvascular abnormalities using computer-assisted intravital microscopy (CAIM) are two objective methods adopted in this laboratory to noninvasively quantify vasculopathy in AD patients. A correlation of increased disease severity with worsened vasculopathy would further bolster a cause and effect relationship. A case report (Case 1) is presented to illustrate the usefulness of following an AD patient with these noninvasive techniques to correlate disease progression with vasculopathy. DESIGN: Patients were selected from a private practice setting who met the Diagnostic and Statistical Manual of Mental Disorders criteria for AD. The Rheolog™, a computer-assisted scanning rheometer, was used to obtain longitudinal measurements of WBV. The microvascular abnormalities in the bulbar conjunctiva were quantified using a severity index (SI, scale 0-15). The patient was observed over a 4 year period from 2005 to 2008. CONCLUSION: This case study shows a correlation of disease progression in an AD patient with worsened vasculopathy. It illustrates the usefulness of WBV and CAIM as tools to quantify vasculopathy in AD patients and additionally suggests a pathogenetic role vasculopathy may play in concert with the amyloid hypothesis.

Comparison of real-time microvascular abnormalities in pediatric and adult sickle cell anemia patients.

The conjunctival microcirculation in 14 pediatric and eight adult sickle cell anemia (SCA) patients was studied using computer-assisted intravital microscopy. The bulbar conjunctiva in SCA patients in both age groups exhibited a blanched/avascular appearance characterized by decreased vascularity. SCA patients from both age groups had many of the same abnormal morphometric [vessel diameter, vessel distribution, morphometry (shape), tortuosity, arteriole:venule (A:V) ratio, and hemosiderin deposits] and dynamic [vessel sludging/sludged flow, boxcar blood (trickled) flow, and abnormal flow velocity] abnormalities. A severity index (SI) was computed to quantify the degree of vasculopathy for comparison between groups. The severity of vasculopathy differed significantly between the pediatric and adult patients (SI: 4.2 ± 1.8 vs. 6.6 ± 2.4; P = 0.028), indicative of a lesser degree of overall severity in the pediatric patients. Specific abnormalities that were less prominent in the pediatric patients included abnormal vessel morphometry and tortuosity. Sludged flow, abnormal vessel distribution, abnormal A:V ratio, and boxcar flow appeared in high prevalence in both age groups. The results indicate that SCA microvascular abnormalities develop in childhood and the severity of vasculopathy likely progresses with age. Intervention and effective treatment/management modalities should target pediatric patients to ameliorate, slow down, or prevent progressive microvascular deterioration.

Correlation of microvascular abnormalities and endothelial dysfunction in Type-1 Diabetes Mellitus (T1DM): a real-time intravital microscopy study.

We hypothesize that real-time in vivo microvascular abnormalities should correlate with biochemical markers of inflammation/endothelial dysfunction in T1DM. Real-time quantification of T1DM and healthy non-diabetic control microcirculation was conducted utilizing computer-assisted intravital microscopy. Selected biochemical markers (high sensitivity C-reactive protein (hsCRP), soluble vascular cell adhesion molecules (sVCAM), soluble intercellular adhesion molecules (sICAM), soluble E-selectin (sE-selectin), nitrotyrosine, superoxide anion (O2-), interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha)) were used for correlation. The severity of microvascular abnormalities, as reflected by the arithmetic severity index (SI), was significantly increased in T1DM vs. controls (5.89 +/- 1.47 vs. 2.34 +/- 1.48; P<0.001). In addition several of the specific microvascular abnormalities (related to flow and morphometry) were significantly more prevalent in the T1DM patients. Finally, the following significant positive correlations existed between the inflammatory/endothelial dysfunction markers and specific microvascular abnormalities: sVCAM and abnormal vessel diameter (P=0.004, OR =1.033, 95% CI for OR =(1.01, 1.056)), superoxide (O2-) release and abnormal vessel distribution (P=0.032, OR =1.798, 95% CI for OR =(1.051, 3.075)), and sE-selectin and abnormal vessel distribution (P=0.036, OR =1.118, 95% CI for OR =(1.007, 1.241)). In view of such significant correlations, we conclude that these specific microvascular abnormalities can serve as unique physiologic markers of endothelial dysfunction to correlate with the biochemical markers of inflammatory/endothelial dysfunction in disease progression and therapeutic efficacy studies.

Treatment of diabetes by transplantation of drug-inducible insulin-producing gut cells.

Most patients with type 1 diabetes rely on multiple daily insulin injections to maintain blood glucose control. However, insulin injections carry the risk of inducing hypoglycemia and do not eliminate diabetic complications. We sought to develop and evaluate a regulatable cell-based system for delivery of insulin to treat diabetes. We generated two intestinal cell lines in which human insulin expression is controlled by mifepristone. Insulin mRNA expression was dependent on the mifepristone dose and incubation time and cells displayed insulin and C-peptide immunoreactivity and glucose-induced insulin release following mifepristone treatment. Cell transplantation followed by mifepristone administration reversed streptozotocin (STZ)-induced diabetes in mice, and this effect was dependent on the mifepristone dose delivered. These data support the notion that engineering regulatable insulin expression within a cell already equipped for regulated secretion may be efficacious for the treatment of insulin-dependent diabetes.

Whole blood viscosity and microvascular abnormalities in Alzheimer's Disease.

We hypothesized that abnormalities in hemorheologic parameters, including vessel diameter, flow velocity, and whole blood viscosity (WBV), would be present in Alzheimer's Disease (AD) and would correlate with microvascular abnormalities (vasculopathy). Using the Rheolog, we obtained WBV profiles, measured at shear rates of 1-1,000 s-1, for 10 AD subjects and age matched non-AD controls. Vessel diameter, flow velocity, and microvascular abnormalities were quantified using computer-assisted intravital microscopy (CAIM) of the conjunctival microcirculation. A Severity Index (SI), scale 0-15, was computed to reflect degree/severity of vasculopathy. AD subjects compared to controls had significantly higher WBV (3.96+/-0.29 cP vs. 3.34+/-0.05 cP, sheared at 300 s-1; P<0.05) and SI (7.00+/-2.36 vs. 0.30+/-0.70; P<0.05). WBV was correlated (rhos=0.648; P<0.05) with SI in AD subjects. These results strongly suggest the simultaneous involvement of hemorheologic abnormalities and systemic vasculopathy in AD.

Pax6 and Pdx1 are required for production of glucose-dependent insulinotropic polypeptide in proglucagon-expressing L cells.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are incretin hormones that play important roles in maintaining glucose homeostasis and are being actively pursued as novel therapeutic agents for diabetes. GIP is produced by dispersed enteroendocrine cells and interestingly at times is coexpressed with GLP-1. We sought to determine the factors that selectively define GIP- vs. GLP-1-expressing cells. We performed comparative immunostaining of Pax6 and Pdx1 in GIP- and GLP-1-secreting cells. We investigated whether Pax6 and Pdx1 activate the human GIP promoter in control IEC-6 cells and GIP-expressing STC-1 cells. EMSA was performed to assess the binding of these transcription factors to the GIP promoter. Pax6 and Pdx1 consistently colocalized in GIP-immunoreactive cells. Cells that coexpress GIP and GLP-1 were Pax6 and Pdx1 positive, whereas cells expressing only GLP-1 were Pax6 positive but did not express Pdx1. GIP promoter activity was enhanced in IEC-6 cells by exogenous Pax6 or Pdx1 and diminished in STC-1 cells by inhibition of endogenous Pax6 or Pdx1 by dominant-negative forms. Promoter truncation analysis revealed a major loss of promoter activity when the sequence between -184 to -145 bp was deleted. EMSA studies indicated that Pax6 and Pdx1 bind to this proximal sequence of the human GIP promoter. Our findings indicate that concomitant expression of Pax6 and Pdx1 is important for GIP expression. Our results also suggest that the presence of Pdx1 defines whether GLP-1-expressing gastrointestinal L cells also coexpress GIP.

Effects of pegylated hamster red blood cells on microcirculation.

The objective of this study was to examine the effects of polyethylene glycol (PEG) treated red blood cells (RBCs) on the microcirculation in a hamster back skin window chamber model. Donor hamster RBCs were PEGylated through an incubation with an activated PEG solution, washed, resuspended, and infused through a 10% volume top loading procedure into the carotid artery in an awake Syrian Golden hamster. Eight hamster groups were treated with activated PEG different sizes and concentrations: 0.05 mM-5 kDa PEG, 0.5 mM-5 kDa PEG, 1.1 mM-5 kDa PEG, 2.2 mM-5 kDa PEG, 22 mM-5 kDa PEG, 0.05 mM-20 kDa PEG, 0.5 mM-20 kDa PEG, and 5 mM-20 kDa PEG. Non-treated RBCs were used as control. The microvascular bed under observation was videotaped 30 min before the infusion and followed for 30 min post infusion. The diameter of individual blood vessels and blood flow velocities in selected vessels was measured. Hematocrit and hemoglobin concentration were recorded before infusion and at the end of experiment. Tissue pO(2) was also monitored. Results showed the hamsters tolerated the PEGylated RBCs without apparent ill effects. No significant changes were recorded for the hematocrit, the hemoglobin concentration, the blood vessel diameters, blood flow velocities, and the interstitial partial oxygen pressure (pO(2)) before, during, and after the injections of PEG-RBCs (P > 0.05). Unlike most hemoglobin-based oxygen carrying compounds, which can cause vasoconstriction, the PEGylated RBCs did not produce any measurable vasoactivity. Together with the absence of rouleaux formation and the fact that PEG molecules can mask the surface antigens on RBCs, PEGylation appeared promising as a circulation enhancement treatment.

Evidence of increased inflammation and microcirculatory abnormalities in patients with type 1 diabetes and their role in microvascular complications.

OBJECTIVE: Type 1 diabetes is associated with increased microvascular complications and inflammation. The monocyte-macrophage is a pivotal cell in atherogenesis. There are scanty data on noninvasive measures of microvascular abnormalities and inflammation in type 1 diabetic subjects with microvascular complications. Thus, we examined systemic and cellular biomarkers of inflammation in type 1 diabetic patients with microvascular complications (T1DM-MV patients) and type 1 diabetic patients without microvascular complications (T1DM patients) compared with matched control subjects and determined the microcirculatory abnormalities in the T1DM and T1DM-MV patients using computer-assisted intravital microscopy (CAIM). RESEARCH DESIGN AND METHODS: Fasting blood, 24-h urine, and CAIM measurements were obtained from the T1DM and T1DM-MV patients and matched control subjects. C-reactive protein, E-selectin, nitrotyrosine, monocyte superoxide, and cytokines were elevated in the T1DM and T1DM-MV patients compared with control subjects (P < 0.01). RESULTS: Severity index, as assessed by CAIM, was significantly increased in the T1DM and T1DM-MV patients compared with the control subjects (P < 0.001). There was a significant increase in C-reactive protein, nitrotyrosine, vascular cell adhesion molecule and monocyte superoxide anion release, and interleukin-1 release in T1DM-MV compared with T1DM patients (P < 0.05). T1DM-MV patients had significantly increased CAIM severity index and microalbumin-to-creatinine ratio compared with T1DM patients (P < 0.05). Furthermore, pp38MAPK, pp65, and pERK activity were significantly increased in monocytes from the T1DM and T1DM-MV patients compared with those from the controls subjects, and pp38MAPK and pp65 activity were significantly increased in the T1DM-MV compared with the T1DM patients (P < 0.01). CONCLUSIONS: T1DM-MV patients have increased inflammation compared with T1DM patients. CAIM provides an effective biomarker of microvascular complications, since it is significantly elevated in T1DM-MV compared with T1DM patients and can be monitored following therapies targeted at improving inflammation and/or microvascular complications of type 1 diabetes.

Effects of low-volume hemoglobin glutamer-200 versus normal saline and arginine vasopressin resuscitation on systemic and skeletal muscle blood flow and oxygenation in a canine hemorrhagic shock model.

OBJECTIVE: To test the hypothesis that low-volume resuscitation with hemoglobin glutamer-200 improves hemodynamic function and tissue oxygenation, whereas arginine vasopressin resuscitation improves blood pressures more than low-volume saline or hemoglobin glutamer infusion but compromises systemic and muscle blood flow and oxygenation. DESIGN: Randomized laboratory investigation. SETTING: University research facility. SUBJECTS: Nineteen dogs. INTERVENTIONS: Dogs were instrumented to determine heart rate; arterial, central venous, pulmonary arterial, and pulmonary arterial occlusion pressures; cardiac output; and quadriceps muscle blood flow and oxygen tension (PMo2). Total and plasma hemoglobin, oxygen content, lactate, pH, standard base excess, and arginine vasopressin levels were determined, and systemic oxygen delivery (Do2I) and extraction ratio were calculated. Measurements were made before and 30 mins following hemorrhage. Dogs were resuscitated over 60 mins with saline (8.5 mL/kg), arginine vasopressin (0.4 IU/kg bolus plus 0.08 IU x kg x min), or 1:1 diluted hemoglobin glutamer-200. Recordings were then repeated. Subsequently, animals received 30 mL/kg shed blood (60 mL x kg x hr), and recordings were repeated immediately and 1 hr later. MEASUREMENTS AND MAIN RESULTS: Hemorrhage ( approximately 52 mL/kg) caused characteristic changes in hemodynamic, hematologic, systemic PMo2, and acid-base variables. Saline resuscitation increased both Do2I and muscle perfusion by 42% and 51%, while arginine vasopressin treatment reduced heart rate by 31% and increased mean arterial pressure by 22% but not cardiac output, Do2I, or muscle blood flow, resulting in a further decrease of PMo2 by 68% and worse metabolic acidosis. Hemoglobin glutamer-200 infusion caused systemic and pulmonary vasoconstriction, however, without deterioration of cardiac output, Do2I, muscle blood flow, or PMo2 despite lack of oxygen content increase. Blood transfusion restored most variables. CONCLUSIONS: Low-volume crystalloid or hemoglobin glutamer-200 resuscitation posthemorrhage may improve (but not restore) macro- and microvascular functions and tissue oxygenation, while arginine vasopressin infusion may only improve blood pressures and result in lower overall systemic perfusion compared with low-volume saline or hemoglobin glutamer-200 treatment and worsening of anaerobic conditions in skeletal muscle.

Comparison of treatment modalities for hemorrhagic shock.

Allogeneic blood resuscitation is the treatment of choice for hemorrhagic shock. When blood is unavailable, plasma expanders, including crystalloids, colloids, and blood substitutes, may be used. Another treatment modality is vasopressin, a vasoconstrictor administered to redistribute blood flow, increase venous return, and maintain adequate cardiac output. While much information exists on systemic function and oxygenation characteristics following treatment with these resuscitants, data on their effects on the microcirculation and correlation of real-time microvascular changes with changes in systemic function and oxygenation in the same animal are lacking. In this study, real-time microvascular changes during hemorrhagic shock treatment were correlated with systemic function and oxygenation changes in a canine hemorrhagic shock model (50-55% total blood loss with a MAP of 45-50 mmHg as a clinical criterion). Following splenectomy and hemorrhage, the dogs were assigned to five resuscitation groups: autologous/shed blood, hemoglobin-based oxygen carrier/Oxyglobin, crystalloid/saline, colloid/Hespan (6% hetastarch), and vasopressin. Systemic function and oxygenation changes were continuously monitored and periodically measured (during various phases of the study) using standard operating room protocols. Computer-assisted intravital video-microscopy was used to objectively analyze and quantify real-time microvascular changes (diameter, red-cell velocity) in the conjunctival microcirculation. Measurements were made during pre-hemorrhagic (baseline), post-hemorrhagic (pre-resuscitation), and post-resuscitation phases of the study. Pre-hemorrhagic microvascular variables were similar in all dogs (venular diameter = 42+/-4 microm, red-cell velocity = 0.55+/-0.5 mm/sec). All dogs showed significant (P < 0.05) post-hemorrhagic microvascular changes: approximately 20% decrease in venular diameter and approximately 30% increase in red-cell velocity, indicative of sympathetic effects arising from substantial blood loss. Microvascular changes correlated with post-hemorrhagic systemic function and oxygenation changes. All resuscitation modalities except vasopressin restored microvascular and systemic function changes close to pre-hemorrhagic values. However, only autologous blood restored oxygenation changes to pre-hemorrhagic levels. Vasopressin treatment resulted in further decreases in venular diameter (approximately 50%) as well as red-cell velocity (approximately 70%) without improving cardiac output. Our results suggested that volume replenishment - not oxygen-carrying capability - played an important role in pre-hospital/en route treatment for hemorrhagic shock. Vasopressin treatment resulted in inadvertent detrimental outcome without the intended benefit.

Improving function and survival of pancreatic islets by endogenous production of glucagon-like peptide 1 (GLP-1).

Glucagon-like peptide 1 (GLP-1) is a hormone that has received significant attention as a therapy for diabetes because of its ability to stimulate insulin biosynthesis and release and to promote growth and survival of insulin-producing beta cells. While GLP-1 is produced from the proglucagon precursor by means of prohormone convertase (PC) 1/3 activity in enteroendocrine L cells, the same precursor is differentially processed by PC2 in pancreatic islet alpha cells to release glucagon, leaving GLP-1 trapped within a larger fragment with no known function. We hypothesized that we could induce GLP-1 production directly within pancreatic islets by means of delivery of PC1/3 and, further, that this intervention would improve the viability and function of islets. Here, we show that adenovirus-mediated expression of PC1/3 in alpha cells increases islet GLP-1 secretion, resulting in improved glucose-stimulated insulin secretion and enhanced survival in response to cytokine treatment. PC1/3 expression in alpha cells also improved performance after islet transplantation in a mouse model of type 1 diabetes, possibly by enhancing nuclear Pdx1 and insulin content of islet beta cells. These results demonstrate a unique strategy for liberating GLP-1 from directly within the target organ and highlight the potential for up-regulating islet GLP-1 production as a means of treating diabetes.

Systemic function, oxygenation and microvascular correlation during treatment of hemorrhagic shock with blood substitutes.

Systemic function and oxygenation changes during hemorrhagic shock treatment were continuously monitored and correlated with real-time microvascular changes. After splenectomy, each dog (n = 12) was hemorrhaged (MAP = approximately 50 mmHg; approximately 40% blood loss = 32-36 ml/kg) and randomly assigned to 4 resuscitation groups: autologous/shed blood, hemoglobin-based oxygen-carrier/Oxyglobin, crystalloid/saline, and colloid/Hespan. Systemic function and oxygenation changes were continuously monitored and measured using standard operating room protocols. Computer-assisted intravital microscopy was used to non-invasively videotape and objectively analyze and quantify real-time microvascular changes in the conjunctival microcirculation. All measurements were made during pre-hemorrhagic (baseline), post-hemorrhagic and post-resuscitation phases of the study. Pre-hemorrhagic microvascular changes were similar in all 12 dogs (venular diameter = 43 +/- 12 microm; red-cell velocity = 0.6 +/- 0.2 mm/s). All dogs showed similar significant (P<0.01) post-hemorrhagic microvascular changes: approximately 20% decrease in venular diameter; approximately 80% increase in red-cell velocity. These microvascular changes correlated with post-hemorrhagic systemic function and oxygenation changes. The resuscitations restored microvascular changes to pre-hemorrhagic values; the microvascular reversals also correlated with post-resuscitation systemic function changes in all groups. However, only shed blood resuscitation restored oxygenation level close to pre-hemorrhagic values. All 12 dogs survived resuscitation treatments despite differences in oxygen-carrying capability between groups.

Harnessing the gut to treat diabetes.

The gut contains one of the largest stem cell populations in the body, yet has been largely overlooked as a source of potentially therapeutic cells. The stem cells reside in the crypts located at the base of the protruding villi, reproduce themselves, and repopulate the gut lining as differentiated cells are sloughed off into the lumen. Some studies have demonstrated that gut stem cells can be isolated and maintained in culture, but the field is currently hampered by the lack of clear markers for these cells. Nevertheless, the relative accessibility of the cells and the similar pathways of differentiation of both intestinal and pancreatic endocrine cells make the gut an attractive potential source of cells to treat diabetes. In particular, it may be possible to recapitulate islet development by the introduction of specific factors to gut stem cells. Alternatively, gut endocrine cells might be coaxed to produce insulin and secrete it into the blood in a meal-responsive manner. Several investigations support the feasibility of both approaches as novel potential therapies for diabetes. Utilizing a patient's own gut cells to re-establish endogenous meal-regulated insulin secretion could represent an attractive approach to ultimately cure diabetes.

Effects of poloxamer 188 treatment on sickle cell vaso-occlusive crisis: computer-assisted intravital microscopy study.

Nine patients with sickle cell disease (SCD) who were hospitalized at UC Davis Medical Center for vaso-occlusive crisis (VOC) were studied as part of a randomized double-blind phase III clinical trial to investigate the real-time effects of poloxamer 188 on VOC. All patients showed significant microvascular changes from normal (steady-state) values during VOC (diminished venular diameter and red cell velocity). The patients were randomly assigned to be treated with poloxamer 188 or placebo. Poloxamer 188 (n = 4) but not placebo (n = 5) significantly reversed these microvascular changes approximately 2 hours postinitiation of loading infusion (100 mg/kg in 1 hour). Further significant improvement induced by poloxamer 188 but not placebo was observed > or = 7 hours postinfusion, resulting in a significant reversal of the microvascular changes to steady-state values.