Congenital erythropoietin over-expression causes "anti-pulmonary hypertensive" structural and functional changes in mice, both in normoxia and hypoxia.

Acute alveolar hypoxia causes pulmonary vasoconstriction that matches lung perfusion to ventilation to optimize gas exchange. Chronic alveolar hypoxia induces pulmonary hypertension, characterized by increased muscularization of the pulmonary vasculature and right ventricular hypertrophy. Elevated erythropoietin (EPO) plasma levels increase hematocrit and blood viscosity and may affect structure and function of the pulmonary circulation. To differentiate between the direct effects of hypoxia and those linked to a hypoxia-induced increase in EPO/hematocrit levels, we investigated the lung vasculature in transgenic mice constitutively over-expressing EPO (termed tg6) upon exposure to normoxia and chronic hypoxia. Despite increased hematocrit levels (approximately 0.86),tg6 mice kept in normoxia did not develop selective right ventricular hypertrophy. The portion of vessels with a diameter of 51-95 microm and >155 microm was increased whereas the portion of small vessels (30-50 microm) was decreased. Pulmonary vascular resistance and the strength of hypoxic vasoconstriction measured in isolated perfused lungs were decreased. Vasoconstrictions induced by the thromboxane mimetic U46619 tended to be reduced. After chronic hypoxia (FiO2 = 0.10, 21 days), vascular resistance and vasoconstrictor responses to acute hypoxia and U46619 were reduced in tg6 mice compared to wildtype controls. Chronic hypoxia increased the degree of pulmonary vascular muscularization in wildtype but not in tg6 mice that already exhibited less muscularization in normoxia. In conclusion, congenital over-expression of EPO exerts an "anti-pulmonary hypertensive" effect, both structurally and functionally, particularly obvious upon chronic hypoxia.

cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue.

Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to approximately 10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs ( approximately 500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from normoxic lungs. Validation by an alternative method was obtained by linking microdissection with real-time polymerase chain reaction (PCR). As suggested by the array data, nine selected genes with different factors of up-regulation were fully confirmed by the PCR technique. Thus, a rapid protocol is presented combining microdissection and array profiling that demands low quantities of initial RNA to assess reliably cell-type-specific gene regulation even within nonneoplastic complex tissues.