Search

Microvascular occlusion in sickle cell disease can be initiated by adhesion of sickle red blood cells (RBCs) to the endothelium. Our objective in this study was to verify the relevance in vivo of our discovery that sickle RBCs adhere abnormally to endothelial P-selectin in vitro. We used computer-assisted intravital microscopy to characterize RBC flow velocity (VRBC) in mice. We found faster VRBC of sickle RBCs in P-selectin knock-out and control mice than in sickle cell mice, which have increased endothelial cell P-selectin expression. Agonist peptide for murine protease-activated receptor-1 (PAR-1), which selectively activates mouse endothelial cells but not platelets, was used to assess the effects of endothelial cell P-selectin on microvascular flow. Suffusion of venules with this agonist stopped flow promptly in normal and sickle mice but not in P-selectin knock-out mice or in control mice pretreated with anti-P-selectin monoclonal antibody or unfractionated heparin (UFH). Agonist-induced slowing of flow was reversed rapidly by suffusion with UFH, provided flow had not already stopped. We conclude that endothelial cell P-selectin contributes to the microcirculatory abnormalities in sickle cell disease and that blocking P-selectin may be useful for preventing painful vasoocclusion in sickle cell disease. (Blood. 2004;104:3378-3385)

Microvascular occlusion in sickle cell disease can be initiated by adhesion of sickle red blood cells (RBCs) to the endothelium. Our objective in this study was to verify the relevance in vivo of our discovery that sickle RBCs adhere abnormally to endothelial P-selectin in vitro. We used computer-assisted intravital microscopy to characterize RBC flow velocity (VRBC) in mice. We found faster VRBCof sickle RBCs in P-selectin knock-out and control mice than in sickle cell mice, which have increased endothelial cell P-selectin expression. Agonist peptide for murine protease-activated receptor-1 (PAR-1), which selectively activates mouse endothelial cells but not platelets, was used to assess the effects of endothelial cell P-selectin on microvascular flow. Suffusion of venules with this agonist stopped flow promptly in normal and sickle mice but not in P-selectin knock-out mice or in control mice pretreated with anti-P-selectin monoclonal antibody or unfractionated heparin (UFH). Agonist-induced slowing of flow was reversed rapidly by suffusion with UFH, provided flow had not already stopped. We conclude that endothelial cell P-selectin contributes to the microcirculatory abnormalities in sickle cell disease and that blocking P-selectin may be useful for preventing painful vasoocclusion in sickle cell disease. (Blood. 2004;104:3378-3385)

The conjunctival microcirculation of 18 homozygous sickle cell disease (SCD) patients during steady-state, painful crisis, and postcrisis conditions was recorded on high-resolution videotapes using intravital microscopy. Selected videotape sequences were subsequently coded, frame-captured, studied, and blindly analyzed using computer-assisted image analysis protocols. At steady-state (baseline), all SCD patients exhibited some of the following morphometric abnormalities: abnormal vessel diameter, comma signs, blood sludging, boxcar blood flow phenomenon, distended vessels, damaged vessels, hemosiderin deposits, vessel tortuosity, and microaneurysms. There was a decrease in vascularity (diminished presence of conjunctival vessels) in SCD patients compared with non-SCD controls, giving the bulbar conjunctiva a “blanched” avascular appearance in most but not all SCD patients during steady-state. Averaged steady-state red cell velocity in SCD patients was slower than in non-SCD controls. During painful crisis, a further decrease in vascularity (caused by flow stoppage in small vessels) and a 36.7% ± 5.2% decrease in large vessel (mostly venular) diameter resulted. In addition, the conjunctival red cell velocities either slowed significantly (6.6% ± 13.1%; P < .01) or were reduced to a trickle (unmeasurable) during crisis. The microvascular changes observed during crisis were transient and reverted to steady-state baseline after resolution of crisis. When combined, intravital microscopy and computer-assisted image analysis (computer-assisted intravital microscopy) represent the availability of a noninvasive tool to quantify microvascular abnormalities in vascular diseases, including sickle cell disease. The ability to identify and relocate the same conjunctival vessels for longitudinal studies uniquely underscores the applicability of this quantitative real-time technology.

The conjunctival microcirculation of 18 homozygous sickle cell disease (SCD) patients during steady-state, painful crisis, and postcrisis conditions was recorded on high-resolution videotapes using intravital microscopy. Selected videotape sequences were subsequently coded, frame-captured, studied, and blindly analyzed using computer-assisted image analysis protocols. At steady-state (baseline), all SCD patients exhibited some of the following morphometric abnormalities: abnormal vessel diameter, comma signs, blood sludging, boxcar blood flow phenomenon, distended vessels, damaged vessels, hemosiderin deposits, vessel tortuosity, and microaneurysms. There was a decrease in vascularity (diminished presence of conjunctival vessels) in SCD patients compared with non-SCD controls, giving the bulbar conjunctiva a “blanched” avascular appearance in most but not all SCD patients during steady-state. Averaged steady-state red cell velocity in SCD patients was slower than in non-SCD controls. During painful crisis, a further decrease in vascularity (caused by flow stoppage in small vessels) and a 36.7% ± 5.2% decrease in large vessel (mostly venular) diameter resulted. In addition, the conjunctival red cell velocities either slowed significantly (6.6% ± 13.1%; P< .01) or were reduced to a trickle (unmeasurable) during crisis. The microvascular changes observed during crisis were transient and reverted to steady-state baseline after resolution of crisis. When combined, intravital microscopy and computer-assisted image analysis (computer-assisted intravital microscopy) represent the availability of a noninvasive tool to quantify microvascular abnormalities in vascular diseases, including sickle cell disease. The ability to identify and relocate the same conjunctival vessels for longitudinal studies uniquely underscores the applicability of this quantitative real-time technology.

The Stroke Prevention Trial has confirmed that utilization of transcranial Doppler ultrasonography (TCD), which examines blood flow in large intracranial vessels, can identify children with sickle cell disease (SCD) who are at high risk of developing a premature stroke. It is not known to what extent the vasculopathy in SCD involves small vessels and whether the abnormalities, if present, correlate with large-vessel vasculopathy. Eighteen children with SCD were examined with TCD to determine middle cerebral artery (MCA) velocity and computer-assisted intravital microscopy (CAIM) to determine bulbar conjunctival vessel velocity during the same visit for vasculopathy correlation. High MCA velocity (≥ 200 cm/sec) was found by TCD in 4 patients who also showed abnormal conjunctival velocity (< 0.2 mm/sec or intermittent trickle flow) by CAIM. Three patients had conditional (≥ 170 cm/sec and < 200 cm/sec) MCA velocity: 2 showed abnormal (trickle) and 1 showed normal conjunctival velocity (1.9 mm/sec). One patient with unmeasurable MCA velocity had abnormal (trickle) conjunctival velocity. Of the remaining 10 patients who had normal MCA velocity, 2 showed abnormal (0.05 mm/sec and 0.1 mm/sec) and 8 showed normal conjunctival velocities (1.1-2.4 mm/sec). The MCA velocities correlated significantly with bulbar conjunctival flow velocities (P ≤ .008, Fisher exact test). A correlation exists between MCA (large-vessel) and conjunctival (small-vessel) flow velocities. CAIM is a noninvasive quantitative technique that might contribute to the identification of SCD patients at high risk of stroke. Small-vessel vasculopathy might be an important pathological indicator and should be further explored in a large-scale study.

The Stroke Prevention Trial has confirmed that utilization of transcranial Doppler ultrasonography (TCD), which examines blood flow in large intracranial vessels, can identify children with sickle cell disease (SCD) who are at high risk of developing a premature stroke. It is not known to what extent the vasculopathy in SCD involves small vessels and whether the abnormalities, if present, correlate with large-vessel vasculopathy. Eighteen children with SCD were examined with TCD to determine middle cerebral artery (MCA) velocity and computer-assisted intravital microscopy (CAIM) to determine bulbar conjunctival vessel velocity during the same visit for vasculopathy correlation. High MCA velocity (≥ 200 cm/sec) was found by TCD in 4 patients who also showed abnormal conjunctival velocity (< 0.2 mm/sec or intermittent trickle flow) by CAIM. Three patients had conditional (≥ 170 cm/sec and < 200 cm/sec) MCA velocity: 2 showed abnormal (trickle) and 1 showed normal conjunctival velocity (1.9 mm/sec). One patient with unmeasurable MCA velocity had abnormal (trickle) conjunctival velocity. Of the remaining 10 patients who had normal MCA velocity, 2 showed abnormal (0.05 mm/sec and 0.1 mm/sec) and 8 showed normal conjunctival velocities (1.1-2.4 mm/sec). The MCA velocities correlated significantly with bulbar conjunctival flow velocities (P≤ .008, Fisher exact test). A correlation exists between MCA (large-vessel) and conjunctival (small-vessel) flow velocities. CAIM is a noninvasive quantitative technique that might contribute to the identification of SCD patients at high risk of stroke. Small-vessel vasculopathy might be an important pathological indicator and should be further explored in a large-scale study.

Abstract 3255

Microvascular abnormalities underlie much of the morbidity observed in sickle cell disease (SCD). Adherence of sickle erythrocytes and leukocytes to the microvascular endothelium leads to vaso-occlusion and end organ ischemia/reperfusion injury. Monocyte chemotactic protein-1 (MCP-1) is a chemokine involved in the recruitment of monocytes to sites of endothelial activation or injury and thus contributes to the pathophysiology of vascular disease. In SCD patients, circulating MCP-1 concentrations are elevated during both steady state and vaso-occlusive crisis. However, the relationship between MCP-1 and microvascular abnormalities in SCD patients has not been assessed. In the present study, we measured a panel of circulating cytokines including serum MCP-1 and hematological biomarkers including serum ferritin in SCD patients who were being monitored by computer-assisted intravital microscopy (CAIM) to assess severity of microvasculopathy.The study included 31 steady state SCD patients (22 females, 9 males) ranging in age from 4–61 years. MCP-1 concentrations were measured by Luminex multiple analyte profiling. Ferritin concentrations were measured by chemiluminometric immunossay. The conjunctival microvasculature was assessed using CAIM. A severity index on a scale of 0–15 was calculated as described previously (Cheung et al, Am J Hematol 85:899, 2010) to quantify the degree of microvasculopathy observed among the patients.The mean ± SD MCP-1 concentration was 445 ± 253 pg/ml (range: 122–1180 pg/ml), mean ± SD ferritin concentration was 1675 ± 1883 ng/ml (range 18–5825 ng/ml), and mean ± SD severity index was 5.0 ± 2.5 (range: 0–9). By multiple regression analysis, with controlling for age and gender, MCP-1 was directly correlated with severity index (p = 0.046), and serum ferritin was directly correlated with MCP-1 (p = 0.019).Monocyte recruitment to the site of endothelial injury involves chemotactic signaling mediated in part by MCP-1, levels of which rise in SCD. Our results indicate that MCP-1 is associated with and may directly contribute to the microvasculopathy observed in SCD patients. Furthermore, we find that MCP-1 levels are correlated with ferritin concentrations. Ferritin reflects body iron stores, and an increase of iron stores in SCD resulting from chronic blood transfusion as well as increased iron absorption associated with hemolysis may thus lead to monocyte/macrophage recruitment to the vascular endothelium and contribute to vasculopathy. Measurements of MCP-1 and ferritin may also serve as surrogate biomarkers of microvasculopathy severity and inflammation.Green: Emisphere - Consultancy : Consultancy; Teva Pharmaceuticals - expert testimony: Consultancy.

Wun: GlycoMimetics: Clinical Trial Sponsorship, Consultancy; Eli Lilly: Clinical Trial Sponsorship, Consultancy. Off Label Use: This drug (GMI-1070) has not been approved for any clinical indication. De Castro:GlycoMimetics: clinical trial sponsorship. Styles:GlycoMimetics: Clinical Trial Sponsorship, Consultancy. Cheung:GlycoMimetics: clinical trial sponsorship. Chase:GlycoMimetics: clinical trial sponsorship. Simon:GlycoMimetics: Research Funding. Magnani:GlycoMimetics: Employment, Equity Ownership. Thackray:GlycoMimetics: Employment, Equity Ownership.

Abstract 262

Novel technologies were used to study the microvascular abnormalities in Type-2 diabetes mellitus (T2DM) patients and to correlate these real-time pathological complications with whole blood viscosity (WBV) over a wide range of shear rates (1–1,000s−1). Computer-assisted intravital microscopy (CAIM) was used to non-invasively and objectively quantify the microvascular abnormalities in the conjunctival microcirculation in T2DM patients (n=12; age range = 45 to 68) and healthy non-diabetic control subjects, with the medical history of each subject blinded to the investigators. Fifteen recognizable microvascular abnormalities existed in T2DM patients and not in the control subjects, though not all the abnormalities were found in each patient. A severity index (SI) -- the arithmetic sum of microvascular abnormalities in each patient quantified via CAIM -- was computed to give a score to correlate with medical history, WBV and shear rates. T2DM SI (6.9±1.7) differed significantly (P<0.01) from control SI (0.6±0.7) and correlated significantly with disease severity, but not with the duration of the disease since diagnosis. The results, together with results from a previous study, lend support to the hypothesis that diabetic complications may have occurred in the pre-diabetic period; indicative of the existence of a “time window” before the onset of clinically detectable hyperglycemia. To adequately assess WBV to correlate with T2DM SI, a computer-assisted scanning capillary viscometer, the Rheolog™, was used to generate a viscosity profile for each patient or control subject over a range of shear rates (1 to 1,000s−1) using one 3-ml citrated venous blood sample from each subject. Based on the viscosity profiles generated, WBV of T2DM patients at various shear rates differed significantly from control values. For example, averaged WBV of T2DM patients at a shear rate of 300s−1 (4.01±0.33cp) differed significantly (P<0.02) from averaged WBV of control subjects (3.34±0.05cp). WBV correlated with SI of T2DM patients, disease severity and medical records. This correlation may have unique clinical implications - it can be used to study shear-stress induced pathogenesis on endothelial dysfunction in various vascular diseases and in early detection of T2DM in carriers (e.g. siblings of T2DM patients) before clinical diagnosis of T2DM during the pre-diabetic “time window” of early detection. Follow-up studies on the correlation between real-time microvascular abnormalities (SI), disease severity, WBV and shear rates in Alzheimer’s Disease patients and in early detection of T2DM are in progress.

Novel technologies were used to study the microvascular abnormalities in Type-2 diabetes mellitus (T2DM) patients and to correlate these real-time pathological complications with whole blood viscosity (WBV) over a wide range of shear rates (1–1,000s−1). Computer-assisted intravital microscopy (CAIM) was used to non-invasively and objectively quantify the microvascular abnormalities in the conjunctival microcirculation in T2DM patients (n=12; age range = 45 to 68) and healthy non-diabetic control subjects, with the medical history of each subject blinded to the investigators. Fifteen recognizable microvascular abnormalities existed in T2DM patients and not in the control subjects, though not all the abnormalities were found in each patient. A severity index (SI) -- the arithmetic sum of microvascular abnormalities in each patient quantified via CAIM -- was computed to give a score to correlate with medical history, WBV and shear rates. T2DM SI (6.9±1.7) differed significantly (P<0.01) from control SI (0.6±0.7) and correlated significantly with disease severity, but not with the duration of the disease since diagnosis. The results, together with results from a previous study, lend support to the hypothesis that diabetic complications may have occurred in the pre-diabetic period; indicative of the existence of a “time window” before the onset of clinically detectable hyperglycemia. To adequately assess WBV to correlate with T2DM SI, a computer-assisted scanning capillary viscometer, the Rheolog™, was used to generate a viscosity profile for each patient or control subject over a range of shear rates (1 to 1,000s−1) using one 3-ml citrated venous blood sample from each subject. Based on the viscosity profiles generated, WBV of T2DM patients at various shear rates differed significantly from control values. For example, averaged WBV of T2DM patients at a shear rate of 300s−1(4.01±0.33cp) differed significantly (P<0.02) from averaged WBV of control subjects (3.34±0.05cp). WBV correlated with SI of T2DM patients, disease severity and medical records. This correlation may have unique clinical implications - it can be used to study shear-stress induced pathogenesis on endothelial dysfunction in various vascular diseases and in early detection of T2DM in carriers (e.g. siblings of T2DM patients) before clinical diagnosis of T2DM during the pre-diabetic “time window” of early detection. Follow-up studies on the correlation between real-time microvascular abnormalities (SI), disease severity, WBV and shear rates in Alzheimer's Disease patients and in early detection of T2DM are in progress.