Endothelium Of Small Arteries Research Articles

Endothelial Hemoglobin Alpha Mediates Iron Regulation of Endothelial Nitric Oxide

Iron deficiency anemia (IDA) is a common comorbidity in cardiovascular disease. Previous clinical studies have found iron deficiency anemia (IDA) increases endothelial NO signaling and attributed this change to the reduction in circulating hemoglobin. However, it has been shown in ex vivo preparations that endothelial NO signaling is unaffected by the presence of erythrocytes suggesting changes in circulating hemoglobin do not alter NO signaling. Our lab has previously shown the hemoglobin-α (Hbα) is expressed in the endothelium of small arteries and scavenges NO. We hypothesized endothelial Hbα, rather than blood hemoglobin is responsible for the increased NO signaling in IDA. We first tested this hypothesis in a mouse model of IDA in which we were able to replete vascular iron stores. Male and female C57BL/6 mice were fed an iron deficient (2-6 ppm Fe) or nutrient matched control diet (48 ppm Fe) beginning at weaning. IDA mice underwent phlebotomies (10% blood volume) at 9 and 11 weeks old. To restore vascular iron stores, a subset of IDA mice received a single i.p. injection of iron dextran (FeDex; 20 mg/kg) at 12 weeks. All measurements were taken one week later. As expected, IDA mice had low blood hemoglobin, and FeDex did not rescue the anemia but did rescue vascular iron stores as measured by ferritin light chain protein. To investigate NO signaling, we used laser speckle contrast imaging to measure changes in in vivo blood flow in response to the NO synthase inhibitor L-NAME. The magnitude of the response to L-NAME is an indicator of NO signaling. IDA mice exhibited an exaggerated response to L-NAME indicating increased NO signaling. FeDex treatment restored the L-NAME response back to control levels suggesting reduced vascular iron stores, rather than the anemia itself increased NO signaling. In order to investigate the role of endothelial Hbα, we repeated these studies in endothelial specific Hbα knockout mice (Hba1fl/fl-Cdh5-CreERT2+). Lack of endothelial Hbα did not prevent the increased response to L-NAME in IDA mice but did prevent the rescue by FeDex. This suggests a model in which endothelial Hba participates in regulating NO in response to iron but not the increased NO in IDA. Because Hbα does not explain the initial increase in NO, we have begun investigating other anemia-associated NO stimulators. Erythropoetin (EPO) is known to stimulate NO production in endothelial cells and is elevated in our anemia model regardless of FeDex treatment. Future work will investigate the role of EPO in regulating NO in IDA. University of Virginia Basic and Translational Research Training Grant T32 007284 (LSD, MAL, SAL); NIH HL088554; LaunchPad; Lipedema Foundation; AHA Predoctoral Fellowship (MAL). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial Hemoglobin Alpha Mediates Iron Regulation of Endothelial Nitric Oxide

Iron deficiency is associated with heart failure, chronic kidney disease, and chronic inflammation. All of these pathologies are associated with endothelial dysfunction and impaired nitric oxide (NO) signaling. Previous clinical studies have found iron deficiency anemia (IDA) increases endothelial NO signaling and attributed this change to the reduction in circulating hemoglobin. However, while free hemoglobin is a potent scavenger of NO, it has been shown in ex vivo preparations that endothelial NO signaling is unaffected by the presence of erythrocytes suggesting changes in circulating hemoglobin do not alter NO signaling. For these reasons we focused on endothelial iron regulation. Using single cell RNA sequencing data of isolated mesenteric and adipose endothelial cells generated by our lab, we have observed arterial endothelial cells have higher transferrin receptor expression compared to capillary, veinous, and lymphatic endothelial cells. This suggests that the arterial endothelium, which controls blood flow and is especially susceptible to endothelial dysfunction, may be particularly sensitive to changes in iron status. Additionally, our lab has previously shown the alpha chain of hemoglobin (Hbα) is expressed in the endothelium of small arteries and scavenges NO. We therefore hypothesized endothelial Hbα, rather than blood hemoglobin is responsible for the increased NO signaling in IDA. We first tested this hypothesis in a mouse model of IDA in which we were able to replete vascular iron stores. C57BL/6 mice were fed an iron deficient (2-6 ppm Fe) or nutrient matched control diet (48 ppm Fe) beginning at weaning. IDA mice underwent phlebotomies (10% blood volume) at 9 and 11 weeks old to aid in the progression of anemia. To restore vascular iron stores, a subset of IDA mice received a single i.p. injection of iron dextran (FeDex; 20 mg/kg) at 12 weeks. One week later complete blood counts were assessed, tissues were collected, and blood flow studies were performed. As expected, IDA mice had low blood hemoglobin (Con: 15.54 ± 0.43 vs. IDA: 6.04 ± 0.35 g/dL; p < 0.0001). FeDex did not rescue the anemia (8.55 ± 0.74 g/dL; p < 0.0001 vs. Con), but did rescue vascular iron stores as measured by ferritin light chain protein. To investigate NO signaling, we used laser speckle contrast imaging to measure changes in in vivo blood flow in response to the NO synthase inhibitor L-NAME. The magnitude of the response to L-NAME is an indicator of NO signaling. IDA mice exhibited an exaggerated response to L-NAME indicating increased NO signaling. Rescuing vascular iron stores with FeDex restored the L-NAME response back to control levels suggesting vascular iron stores, rather than the anemia itself increased NO signaling. In order to investigate the role of endothelial Hbα, we repeated these studies in endothelial specific Hbα knockout mice (Hba1 fl/fl-Cdh5-CreER T2+) developed by us. Lack of endothelial Hbα did not prevent the increased response to L-NAME in IDA mice, but did prevent the rescue by FeDex. These data suggest a model in which endothelial Hbα participates in regulating NO in response to iron, but not the increased NO in IDA.

Pulmonary tumor thrombotic microangiopathy showing aggressive course after transurethral resection of urinary bladder: an autopsy case report

A 77-year-old man developed pulmonary tumor thrombotic microangiopathy (PTTM) 2 days after undergoing transurethral resection for urothelial carcinoma (G3) of the urinary bladder and died of respiratory failure 6 days later. Histological findings demonstrated marked intimal fibrocellular proliferation, fibrin thrombi, and both cancer cells and fibrin thrombi in the arteries of the lungs, findings consistent with PTTM. Prominent stenosis in arteries smaller than 300 μm was also seen. The Ki-67 labeling index of primary and metastasized cancer cells was 62.4 % and 70.2 %, respectively. The membranes of metastasized cancer cells expressed E-cadherin, similar to membranes in the urinary bladder. An aggressive PTTM course is affected by intimal fibrocellular proliferation and the high cell proliferation of cancer cells. Furthermore, prominent stenosis in small arteries and membranous staining of E-cadherin of metastasized cells suggest that cancer cells formed clusters by maintaining adhesion molecules and migrated into the arteries of the lungs, where they easily caused damage to the endothelium of small arteries, in contrast to dispersed cancer cells.

Expression Patterns of Heat Shock Proteins in Lungs of Neonates With Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is associated in many cases with pulmonary hypertension. Currently, extracorporeal membrane oxygenation (ECMO) is one of the possible modalities of treatment of pulmonary hypertension and prevention of parenchymal lung injury in neonates with CDH. Molecular stress is present in the lungs of neonates with CDH. To test this hypothesis, we investigate the expression pattern of stress genes (heat shock proteins [HSPs] 27 and 70) in lungs of patients with CDH who have pulmonary hypertension, and evaluate the influence of ECMO on the expression levels of these genes to understand the underlying molecular mechanisms. Paraffin-embedded lung autopsy specimens from patients with CDH and lung hypoplasia who either did or did not receive ECMO treatment and age-matched controls were immunostained by means of monoclonal antihuman antibodies against HSP 70 and HSP 27, with the streptavidin-biotin complex method. Level III academic children's hospital. Expression levels of both HSP 27 and HSP 70 were semiquantitatively evaluated in bronchial epithelium, as well as in medial smooth muscle cells (SMCs) and endothelium of large and small pulmonary arteries, by means of a score ranging from 0 to 4. Statistical analysis of the data was performed with the nonparametric Mann-Whitney test, with significant probability value at P < or = .05. For HSP 70, the most pronounced immunoreactivity was observed in the bronchial epithelium, followed by the medial SMCs of small arteries (of external diameter < 200 microm). The overall expression was significantly higher in patients with CDH than controls in bronchi as well as in pulmonary arteries. For HSP 27, intense expression was found in medial SMCs, followed by the bronchial epithelium in controls, with significantly increased expression in medial SMCs of large and small arteries in patients with CDH. Treatment with ECMO was associated with significantly reduced expression levels of HSP 70 in medial SMCs of both large and small arteries, whereas HSP 27 expression levels were decreased only in small arteries. In addition, the expression levels of both HSPs were significantly lower in endothelium of small arteries. Increased expression of HSPs in CDH points to a condition of pulmonary stress. This pulmonary stress appears to be partially ameliorated by ECMO treatment. This may point to one of the mechanisms by which ECMO alleviates pulmonary hypertension associated with CDH.

Endothelial function in subcutaneous resistance arteries from elderly hypertensive and normotensive subjects.

1. Previous studies have indicated that younger hypertensive subjects may have abnormal endothelium-dependent relaxation, which could contribute to the elevated peripheral resistance seen in established hypertension. This study was designed to examine the functional behaviour of the endothelium of small arteries from elderly hypertensive and normotensive subjects. 2. Resistance arteries were obtained from gluteal biopsies taken under local anaesthesia in 28 subjects of mean age 70 (range 60-76) years, and studied in an isometric myograph. Eighteen subjects had untreated essential hypertension, and 10 were normotensive. 3. After measurement of the contractile response to noradrenaline, relaxation responses to a variety of endothelium-dependent (acetylcholine and bradykinin) and endothelium-independent (iloprost and sodium nitroprusside) mechanisms were assessed in vessels precontracted with noradrenaline. Endothelium-dependent responses were also studied after incubation with NG-nitro-L-arginine to inhibit nitric oxide synthase. 4. There were no significant differences in the contraction or relaxation responses between elderly subjects with or without high blood pressure. Inhibition of nitric oxide synthase prevented any relaxation with acetylcholine and significantly attenuated the relaxation with bradykinin. Near-complete relaxation was however achieved with the endothelium-independent vasodilator sodium nitroprusside. 5. Hypertension in elderly subjects is not associated with a reduction in endothelial vasodilating function in the subcutaneous vessels of the gluteal region compared with age-matched normotensive controls. The results of this study do not support the hypothesis of a defect of resistance artery endothelium-dependent relaxation in the pathophysiology of hypertension in the elderly.

Monoclonal Antibody Against Bone Marrow Stromal Cells Its Production and Characterization

Bone marrow stromal cells play an essential role in the proliferation and differentiation of hematopoietic stem cells (1, 2). As a means of analyzing of the bone marrow microenvironment immunohistochemically, we attempted to produce a rat monoclonal antibody against the murine preadipocyte line H-1 derived from long-term bone marrow culture (LTBMC) of C57BL/6 mice (3, 4). A newly established monoclonal antibody, designated R4-A9, was obtained from a hybridoma prepared by fusion of Y.B2/3.0Ag20(YO) rat myeloma cells with spleen cells of LEW rats immunized with H-1 cells. The immunofluorescence of live H-1 cells showed that the antigen reacting with this antibody was strongly expressed on the cell surface. The specificity of R4-A9 was assessed immunohistochemically on frozen sections of various tissues from normal adult mice. R4-A9 demonstrated specificity for hematopoietic stroma in bone marrow and spleen. No staining was observed in thymus, lymph nodes or other tissues examined, with the exception of Leydig cells in the testis and the endothelium of small arteries in several organs. Detailed immunohistochemical observations at both the light microscopy and electron microscopy level showed that R4-A9 selectively reacted with the sinusoidal endothelium, perisinusoidal adventitial cells (5) (adventitial reticular cells (6] and intersinusoidal reticular cells (5) and the reticular cells of the splenic red pulp. These findings indicate that reticular cells and the endothelium of the bone marrow possess the common cell surface molecules recognized by R4-A9. SDS-PAGE analysis showed that R4-A9-immunoprecipitated proteins had a molecular mass of 100 kDa under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Localized areas of high alkaline phosphatase activity in endothelium of arteries.

THE earliest reports of histochemical reactions for phosphatases in the alkaline pH-range described activity in the endothelium of capillaries, “sometimes” in the endothelium of “blood vessels” and in the adventitia of medium-sized arterioles1–3. Differences in the activity of this enzyme in capillaries of various organs were noted. Since then, no specific localization of alkaline phosphatase activity has been described in the vascular tree4–6. The purpose of the present communication is to report the presence of intense alkaline phosphatase activity localized in the endothelium of small arteries and arterioles at their origin from larger vessels.