The role of matrix metalloproteinase in the intimal sarcoma-like cells derived from endarterectomized tissues from a chronic thromboembolic pulmonary hypertension patient.

Sarcoma-like cells (SCLs) were derived from endarterectomized tissue of a single chronic thromboembolic pulmonary hypertension (CTEPH) patient during incubation of those thrombi at second passage as described at our previous report. These cells had malignant potential, with an increased expression of matrix metalloproteinase-14 (MMP-14), leading to tumor emboli within pulmonary arteries in in vivo studies. The purpose of this study was to perform a more detailed evaluation of the characteristics of SCLs, and to elucidate the role of the increased expression of MMP-14 expression in the growth and death of these cells. In order to elucidate the characteristics of SCLs and to confirm the protein expression of MMP-14, three-dimentional culture, invasion assays, a Western blot analysis and immunohistochemical studies were performed. To examine the role of MMP-14 in tumorigenesis, the metalloproteinase inhibitor, batimastat, was administered to SCID mice which were subcutaneously injected with SCLs. Those mice were sacrificed on day 14 and the tumor volume was evaluated. A Western blot analysis showed the increased expression of MMP-14 in comparison to the expression in lung adenocarcinoma cells (A549). Immunohistochemistry showed that SCLs were positive for vimentin, MMP-14, MMP-2 and CD44. However, endothelial markers, such as CD31 and von Willebrand factor (vWF), were negative. The in vivo studies demonstrated that batimastat could suppress the growth of the subcutaneous tumors formed by the SCLs. This study suggested that MMPs had critical roles on the pathological activities of SCLs and that batimastat might have anti-proliferative and anti-invasive effects on these cells

Immunohistochemical and Morphometric Assessment on the Biological Function and Vascular Endothelial Cells in the Initial Process of Cortical Porosity in Mice With PTH Administration

To clarify the cellular mechanism of cortical porosity induced by intermittent parathyroid hormone (PTH) administration, we examined the femoral cortical bone of mice that received 40 mu g/kg/day (four times a day) human PTH (hPTH) (1-34). The PTH-driven cortical porosity initiated from the metaphyseal region and chronologically expanded toward the diaphysis. Alkaline phosphatase (ALP)-positive osteoblasts in the control mice covered the cortical surface, and endomucin-positive blood vessels were distant from these osteoblasts. In PTH-administered mice, endomucin-reactive blood vessels with TRAP-positive penetrated the ALP-positive osteoblast layer, invading the cortical bone. Statistically, the distance between endomucin-positive blood vessels and the cortical bone surface abated after PTH administration. Transmission electron microscopic observation demonstrated that vascular endothelial cells often pass through the flattened osteoblast layer and accompanied osteoclasts in the deep region of the cortical bone. The cell layers covering mature osteoblasts thickened with PTH administration and exhibited ALP, alpha-smooth muscle actin (alpha SMA), vascular cell adhesion molecule-1 (VCAM1), and receptor activator of NF-kappa B ligand (RANKL). Within these cell layers, osteoclasts were found near endomucin-reactive blood vessels. In PTH-administered femora, osteocytes secreted Dkk1, a Wnt inhibitor that affects angiogenesis, and blood vessels exhibited plasmalemma vesicle-associated protein, an angiogenic molecule. In summary, endomucin-positive blood vessels, when accompanied by osteoclasts in the ALP/alpha SMA/VCAM1/RANKL-reactive osteoblastic cell layers, invade the cortical bone, potentially due to the action of osteocyte-derived molecules such as DKK1

Immunolocalization of endomucin-reactive blood vessels and a-smooth muscle actin-positive cells in murine nasal conchae

Objectives: Recently, the biological functions of endomucin-positive blood vessels and closely associated aSMA-positive cells in long bones have been highlighted. The surrounding tissues of the flat bones, such as nasal bones covered with mucosa and lamina propria, are different from those of the long bones, indicating the different distributions of endomucin-positive blood vessels and aSMA-reactive cells in nasal bones. This study demonstrates the immunolocalization of endomucin-reactive blood vessels and aSMA-positive cells in the nasal conchae of 3- and 7-week-old mice. Methods: The nasal conchae of 3-week-old and 7-week-old male C57BL/6J mice were used for immunoreaction of endomucin, CD34, PDGFbb, TRAP, and c-kit. Results: While we identified abundant endomucin-reactive blood vessels in the lamina propria neighboring the bone, not all were positive for endomucin. More CD34-reactive cells and small blood vessels were observed in the nasal conchae of 3-week-old mice than in those of 7-week-old mice. Some aSMApositive cells in the nasal conchae surrounded the blood vessels, indicating vascular smooth muscle cells, while other aSMA-immunopositive fibroblastic cells were detected throughout the lamina propria. aSMA-positive cells did not co-localize with c-kit-immunoreactivity, thereby indicating that the aSMApositive cells may be myofibroblasts rather than undifferentiated mesenchymal cells. Conclusions: Unlike long bones, nasal conchae contain endomucin-positive as well as endomucinnegative blood vessels and exhibit numerous aSMA-positive fibroblastic cells throughout the lamina propria neighboring the bone. Apparently, the distribution patterns of endomucin-positive blood vessels and aSMA-positive cells in nasal conchae are different from those in long bones. (c) 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved

Histochemical examination of blood vessels in murine femora with intermittent PTH administration

Objective: To verify the biological effects of parathyroid hormone (PTH) on the blood vessels in the bone, this study aimed to investigate histological alterations in endomucin-positive blood vessels and perivascular cells in murine femora after intermittent PTH administration. For comparison with blood vessels in the bone, we examined the distribution of endomucin-positive blood vessels and surrounding aSMAimmunoreactive perivascular cells in the liver, kidney, and aorta with or without PTH administration. Methods: Six-week-old male C57BL/6J mice received hPTH [1-34] or vehicle for two weeks. All mice were fixed with a paraformaldehyde solution after euthanasia, and the right femora, kidney, liver, and aorta were extracted for immunohistochemical analysis of endomucin, aSMA, ephrinB2, EphB4, and HIF1a. Light microscopic observations of semi-thin sections and transmission electron microscopic (TEM) observations of ultra-thin sections were performed on the left femora. Results: After intermittent PTH administration, aSMA-reactive/ephrinB2-positive stromal cells appeared around endomucin-positive/EphB4-immunoreactive blood vessels in the bone. In addition, intense immunoreactivities of EphB4 and HIF1a were seen in vascular endothelial cells after the PTH treatment. Several stromal cells surrounding PTH-treated blood vessels exhibited well-developed rough endoplasmic reticulum under TEM observations. In contrast to bone tissues, aSMA-positive stromal cells did not increase around the endomucin-positive blood vessels in the kidney, liver, or aorta, even after PTH administration. Conclusion: These findings show that intermittent PTH administration increases aSMA-reactive/ephrinB2-positive perivascular stromal cells in bone tissue but not in the kidney, liver, or aorta, suggesting that PTH preferentially affects blood vessels in the bone. (c) 2022 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology

Batimastat suppressed the growth of SCLs in xenograft models.

<p>Subcutaneous tumors were formed on the backs of the mice in the control group (A) and batimastat group (B). It was revealed that there were significant differences in the tumor weights between the control group (n = 6) and batimastat group (n = 4) (C). (* p-value <0.05, versus control group, by Student's t-test)</p

Batimastat suppressed the biological activities of SCLs in <i>in vivo</i> studies.

<p>(A) Batimastat, a synthetic matrix metalloproteinase inhibitor, suppressed the proliferation of SCLs 72 hours after incubation with batimastat. The number of SCLs that survived following the culture with batimastat was lower than that in the control group. (* p-value <0.05, **p-value<0.01 versus control group, by Student's t-test, n = 6). (B) The invasion assays revealed that the SCLs incubated with batimastat were less invasive than those in the control group. (* p-value <0.05 versus control group, **p-value<0.01 versus control group, by Student's t-test, n = 8)</p

Tumors which grew along the intimal surface of the pulmonary arteries.

<p>(A) A resected lung from a mouse. SCID mice were sacrificed 28 days after the injection of SCLs. Many nodules were located on the surface of the lungs. Arrow heads showed some of these nodules. (B) HE staining showed that the tumors were composed of a central area with necrosis and a peripheral zone filled with SCLs. (C) Immunohistochemical staining for MMP-14 revealed that the peripheral area of tumors was positive for MMP-14, and the central necrotic area was negative for MMP-14. (D) Immunohistochemical staining for MMP-2 showed that MMP-2 was weakly positive in the peripheral zone. (Scale bars show 100 µm.)</p

The results of the immunohistochemical analysis of the SCLs.

<p>The SCLs were positive for vimentin (A), MMP-14 (F), MMP-2 (G) and CD44 (H). In contrast, the staining for α-SMA (B), VWF (C), CD31 (D) and desmin (E) was negative. (Scale bars show 50 µm.)</p

The results of the Western blot analysis.

<p>(A) The Western blot analysis revealed that the MMP-14 protein expression in SCLs was higher than that in A549 cells. There was no significant difference in the protein expression of MMP-14 in SCLs incubated in the media with or without growth factors. (B) Batimastat did not suppress the SCLs' expression of the MMP-14 and MMP-2 proteins. (C)(D) The band signal strength of MMP-14 (c) and MMP-2 (d) expressed as a ratio to beta-actin. No significant differences in the signal strength were recognized 24 hours and 48 hours after batimastat exposure between the batimastat and control groups. (“N.S.” showed no significant difference between two groups analyzed by Student's t-test.)</p