Rat lung tissues were obtained from five adult male Sprague-Dawley rats weighing 200 g. The rats were injected subcutaneously with the synthetic vascular endothelial growth factor (VEGF) receptor 2 inhibitor 3-[(2,4 dimethylpyrrol-5-yl) meth-ylidenyl]-indolin-2-one (SU5416). After a single injection (200 mg/kg) of SU5416, the animals were exposed to 3 weeks of chronic hypoxia (simulated altitude of 5,000 m in a hypobaric chamber, with an inspired partial oxygen pressure of approximately 76 mm Hg). The animals were then returned to Denver altitude. Immunohistochemistry for caveolin 1 and 2 expression was performed as described above.
Decreased Expression of Caveolin 1 and 2 in the Vascular Lesions of Patients With Severe PH Caveolin staining was ubiquitous throughout the lung tissue of all patients with primary and secondary PH and in the liver hemangioma. However, the complex vascular structures of severe PH—the plexiform lesions—frequently demonstrated a striking decrease or absence of caveolin 1 and 2 staining (Fig 1, 2), although some of the cells lining the residual lumens stained positive for caveolin (Fig 2). When serial sections were stained using antibodies against Factor VIII-r.ag, a-SMA, and caveolin 1, some of the plexiform lesions demonstrated composite endothelial and smooth-muscle cell phenotypes. canadian good neighbor pharmacy
In these lesions, both the endothelial and smooth-muscle cells lost their caveolin expression (Fig 3). Altogether, 26 of 29 vascular lesions in the patients with severe (primary and secondary) PH were found to be markedly abnormal, ie, they showed clearly decreased or absent staining for caveolin 1. In the 26 lesions, approximately 70% (range, 52 to 85%) of the cells comprising each plexiform lesion demonstrated lack of caveolin 1 expression (as assessed by Zeiss image analysis). Caveolin 2 expression mirrored the caveolin 1 expression pattern. In contrast, remodeled pulmonary arteries with medial hypertrophy only (Fig 4) and the endothelium of the hemangioma (Fig 5) demonstrated abundant caveolin 1 and 2 expression. The Western blot of the whole lung extract did not demonstrate a significant difference in the expression of caveolin 1 protein (Fig 6), illustrating the importance of immunohistochemical studies for the documentation of localized (ie, lesion) abnormalities. With p < 0.3, we found no significant difference in this comparison.
Figure 1. Top, A: Immunostaining for caveolin 1 demonstrates decreased-to-absent staining in the cells of the plexiform lesion. The endothelial cells Lining the multichanneled lumens are positive (arrows) [original X 400]. Bottom, B: The same lesion as in top, A immunostained for caveolin 2. The caveolin 2 immunostaining mirrors the caveolin 1 immunostaining pattern (original X 400).
Figure 2. Top, A: Plexiform lesion immunostained for caveolin 1. Note that the majority of the cells that make up this complex vascular lesion lack caveolin1 staining and only the endothelial cells that line the multiple lumens are positive (arrows). The surrounding alveolar septal structures express caveolin 1 (arrowheads) [original X 200]. Bottom, B: Plexiform lesion from another patient with PPH demonstrating markedly decreased expression of caveolin 1 (original X 400).
Figure 3. Serial section analysis of a plexiform lesion from a patient with primary PH. The lesion occurs just distal to the bifurcation point of a pulmonary artery. Top, A: The intravascular lesion stains positive for the endothelial cell marker FVIII-r.ag. (arrows) [original X 200]. Center, B: The same lesion as shown in top, A immunostained for a-SMA (arrowhead). The endothelial cells of the plexiform lesion are negative for this smooth-muscle stain (arrows) [original X 200]. Bottom, C: The same lesion as in top, A and center, B immunostained for caveolin 1, showing that both the endothelial as well as the smooth-muscle cells of the plexiform lesion (identified in top, A and center, B above) have decreased or absent caveolin 1 staining (original X 200).
Figure 4. Caveolin 1 expression in alveolar septal cells (arrows), smooth-muscle cells (arrowhead), and endothelial cells (dashed arrow) of remodeled pulmonary hypertensive arteries (original X 200). P = plexiform lesion.
Figure 5. Liver hemangioma showing the high expression of caveolin 1 (arrows) in the endothelial cells. Fibroblast staining is nearly absent (original X 200).
Figure 6. Western blot analysis depicting whole lung extracts from normal lungs (N) as well as primary PH lungs (PPH). No significant difference in caveolin 1 was detected.