Angiogenesis in collagen gels. Angiogenesis in control collagen gels (A) and VEGF (3 μg/ml) containing gels (B) in mouse cranial window was monitored for 14 days. Angiogenesis was quantified as the percentage of squares in the top nylon mesh containing at least one vessel. (A) In the control gels, eNOS^−/−Rag-1^−/− mice (□ KO, n = 10) showed significantly reduced angiogenesis compared with WT Rag-1^−/− (○, n = 10) mice. l-NIL (an iNOS selective inhibitor)-treated Rag-1^−/− mice (▵ WT/l-NIL, n = 4) showed slightly slower angiogenesis than WT/control but the difference was not significant at any time point. (B) In the VEGF gel, l-NIL treatment (▴ WT/l-NIL, n = 8) modestly reduced angiogenesis (P < 0.05 compared with nontreated Rag-1^−/− mice at day 6 and day 8). Angiogenesis in eNOS^−/−Rag-1^−/− mice (■ KO, n = 15) was significantly reduced compared with Rag-1^−/− mice (● WT, n = 16). l-NIL treatment of eNOS^−/−Rag-1^−/− mice (⧫ KO/l-NIL, n = 6) further delayed angiogenesis. (C) The rate of angiogenesis was calculated from time required to fill 50% of squares in the mesh, which is derived from individual angiogenic response curves (A and B). VEGF (3 ng/μl) significantly accelerated angiogenesis in Rag-1^−/− (WT) mice with or without l-NIL treatment, but not in eNOS^−/−Rag-1^−/− (KO) mice. In both control gel and VEGF gel, l-NIL treatment slightly slowed the angiogenesis and KO showed significantly slower angiogenesis than WT mice. Inhibition of both eNOS and iNOS (KO/l-NIL) resulted in further delays in angiogenesis. *, P < 0.05 as compared with WT mice. #, P < 0.05 as compared with WT mice with l-NIL treatment.

Functional vessel density and hemodynamic parameters of angiogenic vessels in VEGF gels. At 14 days after collagen gel implantation, functional vessel density (defined as total length of perfused vessels per unit area), diameter, RBC velocity, and blood flow rate of angiogenic vessels were determined by intravital microscopy. Representative images of angiogenic vessels in VEGF gels (a and b) and VEGF gels with l-NIL treatment (c and d) at 14 days after implantation are shown. The vessel density in eNOS^−/−Rag-1^−/− mice (b and d) was significantly reduced compared with that in Rag-1^−/− mice (a and c). The area of each high-power image is roughly same as the size of one square of the mesh. The autofluorescence of nylon mesh can be seen at the top or left side of a and elsewhere. (Scale bar = 100 μm.) In the VEGF gel, eNOS^−/−Rag-1^−/− mice (KO) showed significantly smaller vascular density compared with Rag-1^−/− (WT) mice with or without l-NIL treatment. l-NIL treatment did not significantly change vascular density in VEGF gels in both WT and KO mice. Vessel diameter showed a trend similar to angiogenesis rate (Fig. 1), WT > WT with l-NIL > KO > KO with l-NIL. However, there was no statistically significant difference (P = 0.0573, WT vs. KO with l-NIL). There was no difference in RBC velocity regardless of mice genotype and treatment. Blood flow rate in individual vessel was reduced in KO mice (P = 0.037) compared with WT mice. A total of 58 locations in 12 WT mice, 30 locations in six l-NIL-treated WT mice, 26 locations in five KO mice, and 15 locations in three l-NIL-treated KO mice were observed. *, P < 0.05 as compared with WT mice. #, P < 0.05 as compared with WT mice with l-NIL treatment.

VEGF-induced vascular permeability in quiescent and angiogenic vessels. Vascular permeability to BSA was determined by intravital microcopy in quiescent (A) and angiogenic (B) vessels. (A) VEGF superfusion significantly increased vascular permeability in C57BL/6 (n = 4) and iNOS^−/− (n = 3) mice but not in eNOS^−/− (n = 3) mice. (B) NOS inhibition in the VEGF gels (Rag-1^−/− (WT) with l-NIL, eNOS^−/−Rag-1^−/− (KO), KO with l-NIL) decreased permeability (1.6 ± 0.3 × 10^-7 cm/s, n = 6, P = 0.0727; 1.2 ± 0.8 × 10^-7 cm/s, n = 3, P = 0.035; 0.5 ± 0.2 × 10^-7 cm/s, n = 3, P = 0.003; respectively) compared with WT mice (2.5 ± 0.4 × 10^-7 cm/s, n = 8). †, P < 0.05 as compared with PBS treatment. *, P < 0.05 as compared with corresponding C57 or WT mice.

NO concentration profiles across VEGF gels. Individual (A and B) and overall averaged (C) measurements of NO concentration profiles across VEGF gels and into the underlying brain tissue. (A and B) The position of the tip relative to the gel surface (Upper) and resulting NO measurement (Lower) as a function of time while the microsensor was advanced are shown for individual NO concentration profiles in VEGF gels implanted in a Rag-1^−/− (WT) mouse (A) and an eNOS^−/−Rag-1^−/− (KO) mouse (B). The peak NO value for this WT mouse (> 4 μM) was located at −362 μm (below the gel surface), near the bottom of the gel. There was no noticeable NO peak in the gel for the KO measurement in the range from 0 to −400 μm. (C) NO profiles averaged over 50 μm increments for a total of 29 profiles in six WT mice, 19 profiles in three l-NIL treated WT mice, 11 profiles in two KO mice, and one profile in one l-NIL-treated KO mouse. Averaged NO profiles demonstrate the pattern WT > WT with l-NIL ≫ KO with and without l-NIL.