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Release of the major excitatory neurotransmitter glutamate by synaptic vesicle exocytosis depends on glutamate loading into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The two principal isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in adult brain that broadly distinguishes cortical (VGLUT1) and subcortical (VGLUT2) systems, and correlates with distinct physiological properties in synapses expressing these isoforms. Differential trafficking of VGLUT1 and 2 has been suggested to underlie their functional diversity. Increasing evidence suggests individual synaptic vesicle proteins use specific sorting signals to engage specialized biochemical mechanisms to regulate their recycling. We observed that VGLUT2 recycles differently in response to high frequency stimulation than VGLUT1. Here we further explore the trafficking of VGLUT2 using a pHluorin-based reporter, VGLUT2-pH. VGLUT2-pH exhibits slower rates of both exocytosis and endocytosis than VGLUT1-pH. VGLUT2-pH recycling is slower than VGLUT1-pH in both hippocampal neurons, which endogenously express mostly VGLUT1, and thalamic neurons, which endogenously express mostly VGLUT2, indicating that protein identity, not synaptic vesicle membrane or neuronal cell type, controls sorting. We characterize sorting signals in the C-terminal dileucine-like motif, which plays a crucial role in VGLUT2 trafficking. Disruption of this motif abolishes synaptic targeting of VGLUT2 and essentially eliminates endocytosis of the transporter. Mutational and biochemical analysis demonstrates that clathrin adaptor proteins (APs) interact with VGLUT2 at the dileucine-like motif. VGLUT2 interacts with AP-2, a well-studied adaptor protein for clathrin mediated endocytosis. In addition, VGLUT2 also interacts with the alternate adaptors, AP-1 and AP-3. VGLUT2 relies on distinct recycling mechanisms from VGLUT1. Abrogation of these differences by pharmacological and molecular inhibition reveals that the

Release of the major excitatory neurotransmitter glutamate by synaptic vesicle exocytosis depends on glutamate loading into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The two principal isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in adult brain that broadly distinguishes cortical (VGLUT1) and subcortical (VGLUT2) systems, and correlates with distinct physiological properties in synapses expressing these isoforms. Differential trafficking of VGLUT1 and 2 has been suggested to underlie their functional diversity. Increasing evidence suggests individual synaptic vesicle proteins use specific sorting signals to engage specialized biochemical mechanisms to regulate their recycling. We observed that VGLUT2 recycles differently in response to high frequency stimulation than VGLUT1. Here we further explore the trafficking of VGLUT2 using a pHluorin-based reporter, VGLUT2-pH. VGLUT2-pH exhibits slower rates of both exocytosis and endocytosis than VGLUT1-pH. VGLUT2-pH recycling is slower than VGLUT1-pH in both hippocampal neurons, which endogenously express mostly VGLUT1, and thalamic neurons, which endogenously express mostly VGLUT2, indicating that protein identity, not synaptic vesicle membrane or neuronal cell type, controls sorting. We characterize sorting signals in the C-terminal dileucine-like motif, which plays a crucial role in VGLUT2 trafficking. Disruption of this motif abolishes synaptic targeting of VGLUT2 and essentially eliminates endocytosis of the transporter. Mutational and biochemical analysis demonstrates that clathrin adaptor proteins (APs) interact with VGLUT2 at the dileucine-like motif. VGLUT2 interacts with AP-2, a well-studied adaptor protein for clathrin mediated endocytosis. In addition, VGLUT2 also interacts with the alternate adaptors, AP-1 and AP-3. VGLUT2 relies on distinct recycling mechanisms from VGLUT1. Abrogation of these differences by pharmacological and molecular inhibition reveals that the

Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2), and a presynaptically localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3) with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Reacidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.

Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2), and a presynaptically localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3) with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Reacidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.

Surface acoustic waves (SAWs) have been used as a rapid and efficient technique for driving microparticles into a three-dimensional scaffold matrix, raising the possibility that SAW may be effective in seeding live cells into scaffolds, that is, if the cells were able to survive the infusion process. Primary osteoblast-like cells were used to specifically address this issue: To investigate the effects of SAW on the cells' viability, proliferation, and differentiation. Fluorescence-labeled osteoblast-like cells were seeded into polycaprolactone scaffolds using the SAW method with a static method as a control. The cell distribution in the scaffold was assessed through image analysis. The cells were far more uniformly driven into the scaffold with the SAW method compared to the control, and the seeding process with SAW was also significantly faster: Cells were delivered into the scaffold in seconds compared to the hour-long process of static seeding. Over 80% of the osteoblast-like cells were found to be viable after being treated with SAW at 20 MHz for 10-30 s with an applied power of 380 mW over a wide range of cell suspension volumes (10-100 mul) and cell densities (1000-8000 cellsmul). After determining the optimal cell seeding parameters, we further found that the treated cells offered the same functionality as untreated cells. Taken together, these results show that the SAW method has significant potential as a practical scaffold cell seeding method for tissue and orthopedic engineering.

Surface acoustic waves (SAWs) have been used as a rapid and efficient technique for driving microparticles into a three-dimensional scaffold matrix, raising the possibility that SAW may be effective in seeding live cells into scaffolds, that is, if the cells were able to survive the infusion process. Primary osteoblast-like cells were used to specifically address this issue: To investigate the effects of SAW on the cells' viability, proliferation, and differentiation. Fluorescence-labeled osteoblast-like cells were seeded into polycaprolactone scaffolds using the SAW method with a static method as a control. The cell distribution in the scaffold was assessed through image analysis. The cells were far more uniformly driven into the scaffold with the SAW method compared to the control, and the seeding process with SAW was also significantly faster: Cells were delivered into the scaffold in seconds compared to the hour-long process of static seeding. Over 80% of the osteoblast-like cells were found to be viable after being treated with SAW at 20 MHz for 10-30 s with an applied power of 380 mW over a wide range of cell suspension volumes (10-100 mul) and cell densities (1000-8000 cellsmul). After determining the optimal cell seeding parameters, we further found that the treated cells offered the same functionality as untreated cells. Taken together, these results show that the SAW method has significant potential as a practical scaffold cell seeding method for tissue and orthopedic engineering.

Spatiotemporal patterns form in many nonlinear physicochemical or biological systems. Although unusual, microfluidic systems are no exception. We observe such patterns to form by colloids along the free surface of a drop beneath which surface acoustic waves propagate, and propose fundamental mechanisms to elucidate their formation. With increasing excitation amplitude, the colloids first assemble into concentric rings and then cluster into islands due to a combination of capillarity and surface acceleration. As the excitation is further increased, fluid streaming commences within the drop, inducing a transient metastable state in which the system alternates between colloidal island formation on the quiescent drop surface and subsequent erasure due to local vortex generation.

Spatiotemporal patterns form in many nonlinear physicochemical or biological systems. Although unusual, microfluidic systems are no exception. We observe such patterns to form by colloids along the free surface of a drop beneath which surface acoustic waves propagate, and propose fundamental mechanisms to elucidate their formation. With increasing excitation amplitude, the colloids first assemble into concentric rings and then cluster into islands due to a combination of capillarity and surface acceleration. As the excitation is further increased, fluid streaming commences within the drop, inducing a transient metastable state in which the system alternates between colloidal island formation on the quiescent drop surface and subsequent erasure due to local vortex generation.

BackgroundThe belief that vascular remodeling and intimal hyperplasia are causes of luminal narrowing in cardiac allograft vasculopathy (CAV) is controversial. This study evaluated the relationship of vascular remodeling and intimal hyperplasia to luminal narrowing 1 year after orthotopic heart transplantation.MethodsIntravascular ultrasound imaging was performed on 190 cardiac transplant recipients at baseline and again 1 year after transplantation as part of a randomized trial of mycophenolate mofetil (MMF) and azathioprine (Aza). Studies 1 year apart were matched at 625 sites. All sites were classified into positive, non-significant and negative remodeling patterns, depending on a change of +/-10% in external elastic membrane area. Of the 190 patients, 99 were randomized to receive MMF, and 91 to receive Aza.ResultsA total of 625 sites were observed. Of these, 52% had no remodeling, 25% exhibited vessel dilation, and 23% had vessel shrinkage in the presence of variable intimal growth (Delta intimal area: 0.73 +/- 1.70 mm2, p < 0.0001; 1.23 +/- 2.02 mm2, p < 0.0001; and 0.20 +/- 1.40 mm2, p = 0.09, respectively). Sixty percent of the lumen loss was due to a decrease in external elastic membrane area and 40% to an increase in intimal area (p = 0.005). Compared with Aza-treated patients, the MMF-treated patients had a lower incidence of vessel shrinkage (17% vs 28%, p = 0.001), and a trend for smaller maximum intimal thickness (0.21 +/- 0.25 mm vs 0.29 +/- 0.31 mm, p = 0.052).ConclusionsPositive remodeling is associated with intimal growth, but negative remodeling does not correlate with any specific change in intimal hyperplasia. Constrictive remodeling is more responsible than intimal hyperplasia for the luminal narrowing that occurs. MMF is more efficacious than azathioprine in preventing the development of CAV at 1 year, by reducing the degree and incidence of vessel shrinkage and the progression of intimal hyperplasia.

BackgroundThe belief that vascular remodeling and intimal hyperplasia are causes of luminal narrowing in cardiac allograft vasculopathy (CAV) is controversial. This study evaluated the relationship of vascular remodeling and intimal hyperplasia to luminal narrowing 1 year after orthotopic heart transplantation.MethodsIntravascular ultrasound imaging was performed on 190 cardiac transplant recipients at baseline and again 1 year after transplantation as part of a randomized trial of mycophenolate mofetil (MMF) and azathioprine (Aza). Studies 1 year apart were matched at 625 sites. All sites were classified into positive, non-significant and negative remodeling patterns, depending on a change of +/-10% in external elastic membrane area. Of the 190 patients, 99 were randomized to receive MMF, and 91 to receive Aza.ResultsA total of 625 sites were observed. Of these, 52% had no remodeling, 25% exhibited vessel dilation, and 23% had vessel shrinkage in the presence of variable intimal growth (Delta intimal area: 0.73 +/- 1.70 mm2, p < 0.0001; 1.23 +/- 2.02 mm2, p < 0.0001; and 0.20 +/- 1.40 mm2, p = 0.09, respectively). Sixty percent of the lumen loss was due to a decrease in external elastic membrane area and 40% to an increase in intimal area (p = 0.005). Compared with Aza-treated patients, the MMF-treated patients had a lower incidence of vessel shrinkage (17% vs 28%, p = 0.001), and a trend for smaller maximum intimal thickness (0.21 +/- 0.25 mm vs 0.29 +/- 0.31 mm, p = 0.052).ConclusionsPositive remodeling is associated with intimal growth, but negative remodeling does not correlate with any specific change in intimal hyperplasia. Constrictive remodeling is more responsible than intimal hyperplasia for the luminal narrowing that occurs. MMF is more efficacious than azathioprine in preventing the development of CAV at 1 year, by reducing the degree and incidence of vessel shrinkage and the progression of intimal hyperplasia.

ObjectivesThis study sought to evaluate the influence of donor lesions on the development of cardiac allograft vasculopathy and outcomes in heart transplant recipients.BackgroundAfter orthotopic heart transplantation (OHT), coronary artery narrowing occurs as a combination of pre-existing donor lesions and new lesions that develop as a result of cardiac allograft vasculopathy.MethodsIntravascular ultrasound (IVUS) studies were performed in 301 recipients at 1.3 +/- 0.6 months and again at 12.2 +/- 0.8 months after OHT. Additional IVUS studies were performed in 90 patients at two and three years of follow-up. Sites at baseline with maximum intimal thickness > or =0.5 mm were defined as pre-existing donor lesions. The angiographic diagnosis of transplant coronary artery disease (TCAD) was defined as a new > or =50% diameter narrowing of a major epicardial vessel.ResultsDonor lesions were present in 30% of the hearts. By IVUS, sites with donor lesions did not have a greater increase in intimal area compared with sites without donor lesions. Angiographically, the incidence of TCAD up to three years after transplantation was higher in recipients with donor lesions than in recipients without donor lesions (25% vs. 4%, p < 0.001). However, the three-year mortality rate was similar between recipients with or without donor lesions (4.5% vs. 5.2%, p = 1.0).ConclusionsPre-existing donor lesions do not act as a nidus for accelerating the progression of intimal hyperplasia. However, patients with donor lesions have a higher incidence of angiographic TCAD. Donor lesions do not affect the long-term survival of patients with OHT up to three years.

ObjectivesThis study sought to evaluate the influence of donor lesions on the development of cardiac allograft vasculopathy and outcomes in heart transplant recipients.BackgroundAfter orthotopic heart transplantation (OHT), coronary artery narrowing occurs as a combination of pre-existing donor lesions and new lesions that develop as a result of cardiac allograft vasculopathy.MethodsIntravascular ultrasound (IVUS) studies were performed in 301 recipients at 1.3 +/- 0.6 months and again at 12.2 +/- 0.8 months after OHT. Additional IVUS studies were performed in 90 patients at two and three years of follow-up. Sites at baseline with maximum intimal thickness > or =0.5 mm were defined as pre-existing donor lesions. The angiographic diagnosis of transplant coronary artery disease (TCAD) was defined as a new > or =50% diameter narrowing of a major epicardial vessel.ResultsDonor lesions were present in 30% of the hearts. By IVUS, sites with donor lesions did not have a greater increase in intimal area compared with sites without donor lesions. Angiographically, the incidence of TCAD up to three years after transplantation was higher in recipients with donor lesions than in recipients without donor lesions (25% vs. 4%, p < 0.001). However, the three-year mortality rate was similar between recipients with or without donor lesions (4.5% vs. 5.2%, p = 1.0).ConclusionsPre-existing donor lesions do not act as a nidus for accelerating the progression of intimal hyperplasia. However, patients with donor lesions have a higher incidence of angiographic TCAD. Donor lesions do not affect the long-term survival of patients with OHT up to three years.