Alkaline Phosphatase Activity Is a Key Determinant of Vascular Responsiveness to Norepinephrine

Here, we tested the hypothesis that TNAP (tissue nonspecific alkaline phosphatase) modulates vascular responsiveness to norepinephrine. In the isolated, Tyrode’s-perfused rat mesentery, 50 µmol/L of L-p-bromotetramisole (L-p-BT; selective TNAP inhibitor, K i =56 µmol/L) significantly reduced TNAP activity and caused a significant 9.0-fold rightward-shift in the norepinephrine concentration versus vasoconstriction relationship. At 100 µmol/L, L-p-BT further reduced mesenteric TNAP activity and caused an additional significant right-shift of the norepinephrine concentration versus vasoconstriction relationship. A higher concentration (200 µmol/L) of L-p-BT had no further effect on either mesenteric TNAP activity or norepinephrine-induced vasoconstriction. L-p-BT did not alter vascular responses to vasopressin, thus ruling-out nonspecific suppression of vascular reactivity. Since in the rat mesenteric vasculature α 1 -adrenoceptors mediate norepinephrine-induced vasoconstriction, these finding indicate that TNAP inhibition selectively interferes with α 1 -adrenoceptor signaling. Additional experiments showed that the effects of TNAP inhibition on norepinephrine-induced vasoconstriction were not mediated by accumulation of pyrophosphate or ATP (TNAP substrates) nor by reduced adenosine levels (TNAP product). TNAP inhibition significantly reduced the Hillslope of the norepinephrine concentration versus vasoconstriction relationship from 1.8±0.2 (consistent with positive cooperativity of α 1 -adrenoceptor signaling) to 1.0±0.1 (no cooperativity). Selective activation of A 1 -adenosine receptors, which are known to participate in coincident signaling with α 1 -adrenoceptors, reversed the suppressive effects of L-p-BT on norepinephrine-induced vasoconstriction. In vivo, L-p-BT administration achieved plasma levels of ≈60 µmol/L and inhibited mesenteric vascular responses to exogenous norepinephrine and sympathetic nerve stimulation. TNAP modulates vascular responses to norepinephrine likely by affecting positive cooperativity of α 1 -adrenoceptor signaling via a mechanism involving A 1 receptor signaling.

Chlorinated lipid induce inflammatory responses in the microcirculation

Previous studies from our research group have shown that chlorinated lipid, generated from the neutrophil-myeloperoxidase (MPO) system, are elevated in a rat sepsis model as well as in plasma of septic patients. Other work reported in multiple experimental studies and human trials have shown that microcirculatory dysfunction is a hallmark of sepsis. However, whether and how chlorinated lipid contribute to microcirculatory dysfunction are still unclear. In the current study, we hypothesized that compared to non-chlorinated lipid, chlorinated lipid could elicit inflammatory responses in the microcirculation. To test this postulate, a specific rat intravital model was developed. Briefly, male Sprague Dawley rats (250-300g) were randomly divided into 4 groups: 2-chloropalmitic acid (2-ClPA) group, 2-chloropalmitaldehyde (2-ClHDA) group, non-chlorinated palmitic acid (PA) group and palmitaldehyde (HDA) group (n=6). Rat mesenteries were exteriorized and superfused with 10 μM of 2-ClPA or 2-ClHDA, respectively. Equimolar concentrations of PA and HDA were applied as controls. Via use of our intravital microscopic approach, the indicators of microcirculatory dysfunction, leukocyte-endothelial interactions (leukocyte rolling and adhesion), mast cell degranulation, reactive oxygen species (ROS) production, and albumin leakage were evaluated at 0 min (baseline), vi 20 min, 50 min and 80 min after the initiation of lipid superfusion, respectively. At the end of experiments, rats were sacrificed and the jejunum was collected. MPO expression from granulocytes of the jejunum was assessed by a fluorescence assay and immunohistochemistry (IHC) staining. Results of this study showed: (1) In the PA group, very few rolling and adhesive leukocytes were detected, while in the 2-ClPA treated group, there was a significant increase in rolling and adhesive leukocytes. At all the time points examined, 2-ClPA produced significant increases in mast cell degranulation, ROS production and albumin leakage, compared with PA. (2) In the HDA group, no increase in leukocyte rolling and adhesion were observed, while 2-ClHDA produced an increase in leukocyte rolling but not stationary adhesion. HDA produced a time-dependent increase in mast cell degranulation, but this was lower than that seen in response to 2-ClHDA. ROS production and albumin leakage were significantly lower in response to HDA than with 2-ClHDA. (3) MPO expression in rat jejunum was almost 2 times higher in 2-ClPA and 2-ClHDA treated groups compared with PA and HDA groups, respectively. Similarly, the IHC staining data showed that MPO expression in 2-ClPA and 2-ClHDA treated groups was more than 3-fold higher than in PA and HDA groups, respectively. In summary, our data indicate that chlorinated lipid, 2-ClPA and 2-ClHDA, elicit inflammatory responses in rat mesentery, which are characterized by elevated leukocyte-endothelial interactions (leukocyte rolling and adhesion), mast cell degranulation, ROS production, and endothelial barrier disruption (albumin leakage). These changes were associated with increased MPO expression level in jejunum submucosa of intestinal segments adjacent to the superfused mesentery in response to the two chlorinated lipid. vii These inflammatory responses mimic those produced by sepsis, thereby providing evidence supporting the hypothesis that chlorinated lipid, generated from the enzymatic activity of neutrophil-MPO system, induce microcirculatory dysfunction via stimulating inflammatory responses when compared to non-chlorinated lipid. In future studies, we propose to directly test this postulate by applying MPO inhibitors to septic models, and monitoring whether chlorinated lipid production would be reduced and the aforementioned inflammatory responses can be attenuated.

Aging is associated with impaired angiogenesis, but normal microvascular network structure, in the rat mesentery

A big problem associated with aging is thought to be impaired microvascular growth or angiogenesis. However, to link the evidence for impaired angiogenesis to microvascular dysfunction in aged tissues, we must compare adult vs. aged microvascular networks in unstimulated scenarios. The objective of this study was to test the hypothesis that aged microvascular networks are characterized by both fewer vessels and the impaired ability to undergo angiogenesis. Mesentery tissues from adult (9-mo) and aged (24-mo) male Fischer 344 rats were harvested and immunolabeled for platelet/endothelial cell adhesion molecule (an endothelial cell marker) according to two scenarios: unstimulated and stimulated. For unstimulated groups, tissues harvested from adult and aged rats were compared. For stimulated groups, tissues were harvested 3 or 10 days after compound 48/80-induced mast cell degranulation stimulation. Unstimulated aged microvascular networks displayed larger mean vascular area per tissue area compared with the unstimulated adult networks. The lack of a decrease in vessel density was supported at the gene expression level with RNA-Seq analysis and with comparison of vessel densities in soleus muscle. Following stimulation, capillary sprouting and vessel density were impaired in aged networks at 3 and 10 days, respectively. Our results suggest that aging associated with impaired angiogenesis mechanisms might not influence normal microvascular function, since unstimulated aged microvascular networks can display a “normal adult-like” vessel density and architecture. NEW & NOTEWORTHY Using a multidimensional approach, we present evidence supporting that aged microvascular networks display vessel density and patterning similar to adult networks despite also being characterized by a decreased capacity to undergo angiogenesis. Thus, vessel loss is not necessarily a characteristic of aging.

Lipopolysaccharide modulates neutrophil recruitment and macrophage polarization on lymphatic vessels and impairs lymphatic function in rat mesentery

Impairment of the lymphatic system is apparent in multiple inflammatory pathologies connected to elevated endotoxins such as LPS. However, the direct mechanisms by which LPS influences the lymphatic contractility are not well understood. We hypothesized that a dynamic modulation of innate immune cell populations in mesentery under inflammatory conditions perturbs tissue cytokine/chemokine homeostasis and subsequently influences lymphatic function. We used rats that were intraperitoneally injected with LPS (10 mg/kg) to determine the changes in the profiles of innate immune cells in the mesentery and in the stretch-mediated contractile responses of isolated lymphatic preparations. Results demonstrated a reduction in the phasic contractile activity of mesenteric lymphatic vessels from LPS-injected rats and a severe impairment of lymphatic pump function and flow. There was a significant reduction in the number of neutrophils and an increase in monocytes/macrophages present on the lymphatic vessels and in the clear mesentery of the LPS group. This population of monocytes and macrophages established a robust M2 phenotype, with the majority showing high expression of CD163 and CD206. Several cytokines and chemoattractants for neutrophils and macrophages were significantly changed in the mesentery of LPS-injected rats. Treatment of lymphatic muscle cells (LMCs) with LPS showed significant changes in the expression of adhesion molecules, VCAM1, ICAM1, CXCR2, and galectin-9. LPS-TLR4-mediated regulation of pAKT, pERK pI-κB, and pMLC20 in LMCs promoted both contractile and inflammatory pathways. Thus, our data provide the first evidence connecting the dynamic changes in innate immune cells on or near the lymphatics and complex cytokine milieu during inflammation with lymphatic dysfunction.