Daniel Goldman

Erythrocytes : Oxygen Sensors and Modulators of Vascular Tone

Through oxygen-dependent release of the vasodilator ATP, the mobile erythrocyte plays a fundamental role in matching microvascular oxygen supply with local tissue oxygen demand. Signal transduction within the erythrocyte and microvessels as well as feedback mechanisms controlling ATP release have been described. Our understanding of the impact of this novel control mechanism will rely on the integration of in vivo experiments and computational models.

Theoretical Models of Microvascular Oxygen Transport to Tissue

To improve understanding of microvascular O2 transport, theoretical modeling has been pursued for many years. The large number of studies in this area attests to the complexities (i.e., biochemical, structural, and hemodynamic) involved. This article focuses on theoretical studies from the last two decades and, in particular, on models of O2 transport to tissue by discrete microvessels. A brief discussion of intravascular O2 transport is first given, highlighting the physiological importance of intravascular resistance to blood‐tissue O2 transfer. This is followed by a description of the Krogh tissue cylinder model of O2 transport by a single capillary, which is shown to remain relevant in modified forms that relax many of the original biophysical assumptions. However, there are many geometric and hemodynamic complexities that require the consideration of microvascular arrays and networks. Multivessel models are discussed that have shown the physiological importance of heterogeneities in vessel spacing, O2 supply, red blood cell flow path, as well as interactions between capillaries and arterioles. These realistic models require sophisticated methods for solving the governing partial differential equations, and a range of solution techniques are described. Finally, the issue of experimental validation of microvascular O2 delivery models is discussed, and new directions in O2 transport modeling are outlined.

T Helper Cell Dysfunction in Systemic Lupus Erythematosus (SLE): Relation to Disease Activity

Patients with systemic lupus erythematosus (SLE) are known to have defects in both humoral and cellular immunity. The significance of defective T cell-mediated immunity and its relationship to disease activity have not been clearly established. We studiedin vitro T helper cell (Th) function in 150 SLE outpatients and correlated Th function with validated measures of disease activity. Interleukin 2 (IL-2) production by peripheral blood mononuclear cells (PBMC) was measured after stimulation with the recall antigens influenza A virus (FLU) and tetanus toxoid (TET), irradiated allogeneic peripheral blood mononuclear cells (ALLO), and phytohemagglutinin (PHA). We observed three patterns of Th response: (1) 76 of 150 (50%) of patients responded to the recall antigens FLU and/or TET, ALLO, and PHA; (2) 62 of 150 (42%) of patients did not respond to recall antigens but responded to ALLO and PHA; and (3) 12 of 150 (8%) of patients did not respond to either recall antigens or ALLO antigens. This diminished T cell function was correlated with higher disease activity as measured by four scales of clinical activity, such that individuals who exhibited morein vitro immune dysfunction presented with significant increases in their clinical activity indicies. The alterations in T cell function could not be accounted for by medication doses alone. Thus, SLE patients have multiple distinct defects at the level of the Th cell which are associated with clinical measures of disease activity.

Acellular hemoglobin-mediated oxidative stress toward endothelium: a role for ferryl iron

We tested the hypothesis that chemical modifications used to produce stable, oxygen-carrying, Hb-based blood substitutes can induce cytotoxicity in endothelial cells in culture because of altered redox activity. We examined the interaction of hydrogen peroxide with nonmodified hemoglobin (HbA0) and two chemically modified hemoglobins, α-cross-linked hemoglobin (α-DBBF) and its polymerized form (poly-α-DBBF). Hydrogen peroxide-induced cell death (as assessed by lactate dehydrogenase release) in bovine aortic endothelial cells (BAEC) was completely inhibited by all three hemoglobin preparations, consistent with their known pseudoperoxidase activity [hemoglobin consumes peroxide as it cycles between ferric (Fe3+) and ferryl (Fe4+) hemes]. However, reaction of the modified hemoglobins, but not HbA0, with hydrogen peroxide induced apoptotic cell death (as assessed by morphological changes and DNA fragmentation) that correlated with the formation of a long-lived ferrylhemoglobin. A preparation of ferryl-α-DBBF free of residual peroxide rapidly induced morphological changes and DNA fragmentation in BAEC, indicative of apoptotic cell death. Redox cycling of chemically modified hemoglobins by peroxide yielded a persistent ferryl iron that was cytotoxic to endothelial cells.We tested the hypothesis that chemical modifications used to produce stable, oxygen-carrying, Hb-based blood substitutes can induce cytotoxicity in endothelial cells in culture because of altered redox activity. We examined the interaction of hydrogen peroxide with nonmodified hemoglobin (HbA0) and two chemically modified hemoglobins, alpha-cross-linked hemoglobin (alpha-DBBF) and its polymerized form (poly-alpha-DBBF). Hydrogen peroxide-induced cell death (as assessed by lactate dehydrogenase release) in bovine aortic endothelial cells (BAEC) was completely inhibited by all three hemoglobin preparations, consistent with their known pseudoperoxidase activity [hemoglobin consumes peroxide as it cycles between ferric (Fe3+) and ferryl (Fe4+) hemes]. However, reaction of the modified hemoglobins, but not HbA0, with hydrogen peroxide induced apoptotic cell death (as assessed by morphological changes and DNA fragmentation) that correlated with the formation of a long-lived ferrylhemoglobin. A preparation of ferryl-alpha-DBBF free of residual peroxide rapidly induced morphological changes and DNA fragmentation in BAEC, indicative of apoptotic cell death. Redox cycling of chemically modified hemoglobins by peroxide yielded a persistent ferryl iron that was cytotoxic to endothelial cells.

Calculations of oxygen transport by red blood cells and hemoglobin solutions in capillaries

A theoretical model is developed to investigate the influence of hemoglobin-based oxygen carriers (HBOCs) on oxygen transport in capillary-size vessels. A discrete cell model is presented with red blood cells (RBCs) represented in their realistic parachute shape flowing in a single file through a capillary. The model includes the free and Hb-facilitated transport of O2 and Hb–O2 kinetics in the RBC and plasma, diffusion of free O2 in the suspending phase, capillary wall, interstitium and tissue. A constant tissue consumption rate is specified that drives the simultaneous release of O2 from RBC and plasma as the cells traverse the capillary. The model mainly focuses on low capillary hematocrits and studies the effect of free hemoglobin affinity, cooperativity and concentration. The results are expressed in the form of cell and capillary mass transfer coefficients, or inverse transport resistances, that relate the spatially averaged flux of O2 coming out of the RBC and capillary to a driving force for O2 diffusion. The results show that HBOCs at a concentration of 7 g/dl reduce the intracapillary transport resistance by as much as 60% when capillary hematocrit is 0.2. HBOCs with high O2 affinity unload most O2 at the venular end, while those with low affinity supply O2 at the arteriolar end. A higher cooperativity did not favor O2 delivery due to the large variation in the mass transfer coefficient values during O2 unloading. The mass transfer coefficients obtained will be used in simulations of O2 transport in complex capillary networks.

The Hopkins Lupus Pregnancy Center: 1987–1991 Update

ABSTRACT: The course of pregnancy in patients with systemic lupus erythematosus is not known. The Hopkins Lupus Pregnancy Center has followed 64 patients (74 pregnancies) prospectively since 1987. Patients are seen monthly and clinical and pregnancy‐related data collected, with particular emphasis on the occurrence of lupus flare. Flare rate during pregnancy was 1.63 per person‐year, compared to 0.64‐0.65 after delivery or in non‐pregnant patients. Flare did not influence pregnancy outcome. Low serum C3 or C4 and high anticardiolipin antibody predicted pregnancy loss, and prednisone dose, aspirin use, diastolic second trimester blood pressure, C3 at first visit, and race predicted preterm birth. Maternal flare and preterm birth are important risks in lupus pregnancy. The latter can be predicted from maternal pregnancy data.

Identification of L- and T-type Ca2+ channels in rat cerebral arteries: role in myogenic tone development

L-type Ca(2+) channels are broadly expressed in arterial smooth muscle cells, and their voltage-dependent properties are important in tone development. Recent studies have noted that these Ca(2+) channels are not singularly expressed in vascular tissue and that other subtypes are likely present. In this study, we ascertained which voltage-gated Ca(2+) channels are expressed in rat cerebral arterial smooth muscle and determined their contribution to the myogenic response. mRNA analysis revealed that the α(1)-subunit of L-type (Ca(v)1.2) and T-type (Ca(v)3.1 and Ca(v)3.2) Ca(2+) channels are present in isolated smooth muscle cells. Western blot analysis subsequently confirmed protein expression in whole arteries. With the use of patch clamp electrophysiology, nifedipine-sensitive and -insensitive Ba(2+) currents were isolated and each were shown to retain electrical characteristics consistent with L- and T-type Ca(2+) channels. The nifedipine-insensitive Ba(2+) current was blocked by mibefradil, kurtoxin, and efonidpine, T-type Ca(2+) channel inhibitors. Pressure myography revealed that L-type Ca(2+) channel inhibition reduced tone at 20 and 80 mmHg, with the greatest effect at high pressure when the vessel is depolarized. In comparison, the effect of T-type Ca(2+) channel blockade on myogenic tone was more limited, with their greatest effect at low pressure where vessels are hyperpolarized. Blood flow modeling revealed that the vasomotor responses induced by T-type Ca(2+) blockade could alter arterial flow by ∼20-50%. Overall, our findings indicate that L- and T-type Ca(2+) channels are expressed in cerebral arterial smooth muscle and can be electrically isolated from one another. Both conductances contribute to myogenic tone, although their overall contribution is unequal.

Biological effects of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil☆

The synthesis of large quantities of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) relative to 1-alkyl-2-acetyl-GPC (PAF; platelet-activating factor) has been demonstrated in several inflammatory cells. The present study has examined agonist and antagonist activities of 1-acyl-2-acetyl-GPC in the human neutrophil. 1-Acyl-2-acetyl-GPC induced a rapid increase in cytosolic calcium in the neutrophil; this effect was detected at 2 x 10(-9) M and was maximal at 10(-6) M. The peak response induced by 1-acyl-2-acetyl-GPC was similar to that induced by PAF although the potency of 1-acyl-2-acetyl-GPC was 300-fold lower than that of PAF. The dose response curves for both 1-acyl-2-acetyl-GPC and PAF were shifted in a parallel fashion by L-652,731 (10(-6) M), a PAF receptor antagonist, suggesting that both 1-acyl-2-acetyl-GPC and PAF act on the same receptor. High concentrations of 1-acyl-2-acetyl-GPC (10(-5) M) induced the release of beta-glucuronidase and lysozyme from the human neutrophil. The percent release of lysozyme induced by 1-acyl-2-acetyl-GPC was consistently higher than that of beta-glucuronidase. Prior stimulation of neutrophils with 1-acyl-2-acetyl-GPC dose-dependently inhibited the increase in cytosolic calcium induced by a subsequent challenge with an optimal concentration of PAF. Similarly, preincubation of neutrophils with 1-acyl-2-acetyl-GPC dose-dependently inhibited beta-glucuronidase and lysozyme release induced by a subsequent stimulation with PAF. The inhibitory effect on degranulation could not be surmounted even by concentrations of PAF 10-fold higher than that of 1-acyl-2-acetyl-GPC. The inhibition appeared to be selective for PAF since 1-acyl-2-acetyl-GPC did not affect f-met peptide-induced degranulation. This study suggests that 1-acyl-2-acetyl-GPC may act as a naturally-occurring specific inhibitor of PAF-induced activation of the human neutrophil.

3' polymorphisms of ETS1 are associated with different clinical phenotypes in SLE

A microsatellite repeat polymorphism was identified in the 3′ flanking region of the human ETS1 gene. Sequencing revealed two CA repeat segments in close proximity. Seven different alleles comprising various combinations of CA repeat units were identified in a healthy control population. Because ETS1 plays a role in lymphocyte development and function, apoptosis, and inflammation, we examined whether any of these polymorphisms were associated with a systemic inflammatory condition, systemic lupus erythematosus (SLE). Inheritance of this disease is polygenic and a recent genome‐wide screen for SLE susceptibility loci revealed linkage with chromosome 11q14‐23, the region in which the ETS1 gene lies. This region has also been identified as a general autoimmune susceptibility region. None of the seven distinct ETS1 alleles appeared statistically more frequently in SLE patients than controls, however, two alleles were associated with particular clinical manifestations. Allele 1 is associated with discoid lesions and allele 7 is associated with vasculitis. While this polymorphism does not directly affect the coding region of ETS1, it may be a marker for overexpression of a particular isoform or inheritance of another polymorphism which does affect function. These data suggest that ETS1 may be involved in the phenotypic expression of systemic lupus erythematosus. Hum Mutat 16:49–53, 2000.

Computational Modeling of Oxygen Transport from Complex Capillary Networks

Microvascular networks, even in skeletal muscle, are highly complex structures, and their function in oxygen transport remains to be addressed completely. In order to do this, a combination of detailed experiments and biophysically-detailed modeling is needed. In the past ten years, there have been several computational studies of the role of non-uniform geometry in oxygen transport in skeletal muscle. Models that allow a significant degree of geometric realism include those of Groebe (1990), Secomb and Hsu (1994), and Hoofd and Turek (1996). To continue investigating the role of physiologically realistic complexity, we have developed a computational model that is general enough to directly utilize detailed geometric, biophysical, and hemodynamic data in oxygen transport calculations.