Hypoxia may act through mechanisms similar to those found in the lung to induce pulmonary vasoconstrictions under acute hypoxic stress; a more chronic stress allows a HIF-induced modulation of response that also affects blood flow and EPO expression.

A, The normal mouse epidermis is hypoxic, based on EF5 binding (red), and expresses HIF-1α protein (red). B, K14 cre recombinase transgene expression was verified by β-galactosidase expression in epidermis in the ROSA25 reporter strain; deletion efficiency of the loxP-flanked allele in the epidermis and hair follicle was 70%. C, EPO protein in the plasma following hypoxic exposure for 14 hours at 9% oxygen is significantly reduced in mice lacking HIF-1α in the epidermis (wt n = 25, K14cre-HIF-1α+^f/+^f n = 11). D, Renal EPO mRNA expression is reduced to normoxic levels in hypoxic K14cre-HIF-1α+^f/+^f mice (wt n = 6, K14cre-HIF-1α+^f/+^f n = 3). All graphs represent mean ± SEM.

A, Special chambers were constructed to isolate inhaled oxygen concentration from the oxygen concentration exposed to the skin. B, In mice breathing 21% O₂, skin hypoxia was not enough to induce increased EPO synthesis. However, in mice breathing 10% O₂, plasma EPO levels were significantly elevated when skin was normoxic (Respired 21% O₂ Skin 21% O₂ n = 2, Respired 21% O₂ Skin 10% O₂ n = 3, Respired 10% O₂ Skin 10% O₂ n = 8, Respired 10% O₂ Skin 21% O₂ n = 9); mice exposed to differential gases for 5 hours. C, Renal EPO expression was higher in mice breathing 10% O₂ while exposed to 21% O₂ (Respired 10% O₂ Skin 10% O₂ n = 6, Respired 10% O₂ Skin 21% O₂ n = 7); mice exposed to differential gases for 5 hours. D, Blood flow is shifted from the skin to the kidney and liver when the skin is hypoxic for 1 hour. This shift is absent when the skin is exposed to normoxia (Respired 10% O₂ Skin 10% O₂ n = 7, Respired 10% O₂ Skin 21% O₂ n = 8). All graphs represent mean ± SEM.

A, Upon deletion of VHL in the epidermis, HIF-1α protein (red) is stabilized and HIF target gene expression is increased in the skin (for each graph wt n = 3, K14cre-VHL+^f/+^f n = 3, for RNA isolation). B, Constitutive or Tamoxifen-induced epidermal deletion of VHL results in highly elevated plasma EPO (wt n = 36, K14cre-VHL+^f/+^f n = 23, K14TAMcre-VHL+^f/+^f n = 5). C, In the K14cre-VHL+^f/+^f mouse, EPO mRNA expression is suppressed in the kidney but increased in the liver. In the tamoxifen inducible K14TAMcre-VHL+^f/+^f, where deletion occurs in the adult, EPO expression is increased in the kidney, and unaffected in the liver (wt n = 9, K14cre-VHL+^f/+^f n = 6, K14TAMcre-VHL+^f/+^f n = 5). D, As blood hematocrit levels increase in the constitutively deleted K14cre-VHL+^f/+^f mice (wt n = 43, K14cre-VHL+^f/+^f 1.5 wks n = 3, K14cre-VHL+^f/+^f 10 wks n = 32), renal EPO mRNA expression is suppressed and hepatic EPO increases, indicating that hematocrit can selectively affect renal EPO expression (wt n = 9, K14cre-VHL+^f/+^f 1.5 wks n = 3, K14cre-VHL+^f/+^f 10 wks n = 6). All graphs represent mean ± SEM.

A, Deletion of HIF-2α but not HIF-1α in the K14cre-VHL+^f/+^f background restores plasma EPO (wt n = 36, K14cre-VHL+^f/+^f n = 23, K14cre-VHL+^f/+^fHIF-1α+^f/+^f n = 15, K14cre-VHL+^f/+^f HIF-2α+^f/+^f n = 3, K14cre-VHL+^f/+^f HIF-1α+^f/+^{f f} HIF-2α+^f/+^f n = 3) and blood hematocrit (B) to wild type levels (wt n = 43, K14cre-VHL+^f/+^f n = 32, K14cre-VHL+^f/+^f HIF-1α+^f/+^f n = 17, K14cre-VHL+^f/+^f HIF-2α+^f/+^f n = 3, K14cre-VHL+^f/+^f HIF-1α+^f/+^{f f} HIF-2α+^f/+^f n = 3). Deletion of both HIF-1α and HIF-2α is similar in effect to deletion of HIF-2α alone. C, Blood oxygen saturation is normal in K14cre-VHL+^f/+^f animals (wt n = 7, K14cre-VHL+^f/+^f n = 6), but animals are hypotensive (wt n = 17, K14cre-VHL+^f/+^f n = 10). D, Blood flow in the K14cre-VHL+^f/+^f is shifted away from the liver and kidney, and toward the skin, as measured by microsphere distribution (wt n = 11, K14cre-VHL+^f/+^f n = 4). E, The shift in blood flow corresponds to increased EF5 binding/hypoxia in the kidney and liver of K14cre-VHL+^f/+^f (wt n = 8, K14cre-VHL+^f/+^f n = 4). F, Nitric oxide metabolites are increased in K14cre-VHL+^f/+^f plasma, demonstrating increased NO production (wt n = 33, K14cre-VHL+^f/+^f n = 8). All graphs represent mean ± SEM.

A, Systemic inhibition of NO synthesis by L-NAME increases plasma EPO in wild type mice (wt n = 36, wt + L-NAME n = 12). Global iNOS knockout mice show significantly elevated EPO plasma levels following hypoxia relative to wild type mice (wt n = 25, iNOS−/− n = 7). B, NO synthesis inhibition by treatment with L-NAME restores plasma EPO levels to levels similar to L-NAME-treated wild type mice, when administered to K14cre-VHL+^f/+^f mice (K14cre-VHL+^f/+^f n = 23, K14cre-VHL+^f/+^f + L-NAME 3 days n = 3, K14cre-VHL+^f/+^f + L-NAME 4 days n = 4). C, NO donor (nitroglycerine) applied to the skin of wild type mice increases plasma EPO levels; similar systemic doses of NO donor do not significantly affect plasma EPO levels (control n = 20, Systemic nitroglycerine n = 3, Epidermal nitroglycerine n = 9). D, Epidermal NO donor administration induces renal EPO mRNA expression (control n = 9, Epidermal nitroglycerine n = 3). E, Epidermal nitroglycerin shifts blood flow (control n = 11, Epidermal nitroglycerine n = 6) towards the skin and increases renal and hepatic hypoxia (F), in a manner similar to that seen in K14cre-VHL+^f/+^f mice (control n = 8, Epidermal nitroglycerine n = 5). All graphs represent mean ± SEM.