Delimiting CD34+ Stromal Cells/Telocytes Are Resident Mesenchymal Cells That Participate in Neovessel Formation in Skin Kaposi Sarcoma

Kaposi sarcoma (KS) is an angioproliferative lesion in which two main KS cell sources are currently sustained: endothelial cells (ECs) and mesenchymal/stromal cells. Our objective is to establish the tissue location, characteristics and transdifferentiation steps to the KS cells of the latter. For this purpose, we studied specimens of 49 cases of cutaneous KS using immunochemistry and confocal and electron microscopy. The results showed that delimiting CD34+ stromal cells/Telocytes (CD34+SCs/TCs) in the external layer of the pre-existing blood vessels and around skin appendages form small convergent lumens, express markers for ECs of blood and lymphatic vessels, share ultrastructural characteristics with ECs and participate in the origin of two main types of neovessels, the evolution of which gives rise to lymphangiomatous or spindle-cell patterns—the substrate of the main KS histopathological variants. Intraluminal folds and pillars (papillae) are formed in the neovessels, which suggests they increase by vessel splitting (intussusceptive angiogenesis and intussusceptive lymphangiogenesis). In conclusion, delimiting CD34+SCs/TCs are mesenchymal/stromal cells that can transdifferentiate into KS ECs, participating in the formation of two types of neovessels. The subsequent growth of the latter involves intussusceptive mechanisms, originating several KS variants. These findings are of histogenic, clinical and therapeutic interest.


Introduction
Kaposi sarcoma (KS) is a vascular tumor of variable behavior that can affect different organs, with a higher incidence in the skin. Since it was first described by Kaposi in 1872 [1], several cell origins have been proposed for KS. It is generally considered that, in its early stages, KS is a reactive angioproliferative lesion with the possibility of evolving into true neoplasia [2][3][4][5] and that the two main cell origins are endothelial cells (ECs) and mesenchymal/stromal cells. The endothelial origin, above all lymphatic endothelium, is based on the KS cell expression for ECs of both lymphatic and blood vessel markers [6,7]. The mesenchymal/stromal cell origin is based on (a) the transdifferentiation demonstrated in other conditions of stromal cells/fibroblasts toward ECs, which can occur directly or be reprogrammed through specific transcription factors [8][9][10][11][12][13], and (b) several studies, including immunologic and genetic observations, which contribute a specific mesenchymal-to-endothelial transition in KS associated with herpesvirus infection [14][15][16][17]. KS herpesvirus-induced EC reprogramming supports viral persistence and contributes to KS tumorigenesis. However, the precise location and type of transforming iting CD34+SCs/TCs on the formation of neovessels and their behavior in the evolutionary stages of KS.

General Characteristics of KS in Patch, Plaque and Nodular Stages
In the patch stage, edema, mild to moderate inflammatory infiltrate, and newly formed vessels could be observed by conventional techniques with hematoxylin-eosin staining ( Figure 1). The perivascular inflammatory infiltrate ( Figure 1A) was composed of mononuclear cells, predominantly lymphocytes and plasma cells, and varying numbers of macrophages. The newly formed vessels were mainly located around the pre-existing blood vessels, with or without perivascular inflammatory infiltrate ( Figure 1B). Newly formed vessels were also present around skin appendages and occasionally in the intervening dermis. Protrusion of pre-existing blood vessels, with or without perivascular inflammatory infiltrate, into the lumen of neovessels gave rise to the promontory sign ( Figure 1C). Red blood cell extravasation occurs with variable intensity, and siderophages can be seen.
general characteristics of the main evolutionary stages of KS; b) the location and characteristics of the CD34+SCs/TCs in the outer layer of pre-existing blood vessels and around skin appendages, with or without perivascular inflammatory infiltrate, and with or without formation of neovessels; and c) the findings supporting the possible role of delimiting CD34+SCs/TCs on the formation of neovessels and their behavior in the evolutionary stages of KS.

General Characteristics of KS in Patch, Plaque and Nodular Stages
In the patch stage, edema, mild to moderate inflammatory infiltrate, and newly formed vessels could be observed by conventional techniques with hematoxylin-eosin staining ( Figure 1). The perivascular inflammatory infiltrate ( Figure 1A) was composed of mononuclear cells, predominantly lymphocytes and plasma cells, and varying numbers of macrophages. The newly formed vessels were mainly located around the pre-existing blood vessels, with or without perivascular inflammatory infiltrate ( Figure 1B). Newly formed vessels were also present around skin appendages and occasionally in the intervening dermis. Protrusion of pre-existing blood vessels, with or without perivascular inflammatory infiltrate, into the lumen of neovessels gave rise to the promontory sign (Figure 1C). Red blood cell extravasation occurs with variable intensity, and siderophages can be seen. The plaque stage revealed an increase in the events of the patch stage, with greater cellularity and an inflammatory infiltrate presenting plasma cells, lymphocytes, and varying numbers of siderophages. The vascular component was more extensive and diffuse than in the patch stage and was also made up of pre-existing and newly formed vessels. The latter appeared as dissecting vascular spaces, with or without the promontory sign, or as fascicles formed by highly cellular narrow cords showing virtual, empty or erythrocyteoccupied lumen (Figure 2A). The cells in the narrow cords showed a spindle-shaped morphology with sparse mitotic figures and without nuclear pleomorphism. Red blood cell extravasation with the presence of siderophages, intracytoplasmic incorporation of red blood cells by ECs (autolumination phenomenon), and intra or extracellular hyaline globules, probably corresponding to modified red blood cells, was observed with relative frequency ( Figure 2B,C). The plaque stage revealed an increase in the events of the patch stage, with greater cellularity and an inflammatory infiltrate presenting plasma cells, lymphocytes, and varying numbers of siderophages. The vascular component was more extensive and diffuse than in the patch stage and was also made up of pre-existing and newly formed vessels. The latter appeared as dissecting vascular spaces, with or without the promontory sign, or as fascicles formed by highly cellular narrow cords showing virtual, empty or erythrocyteoccupied lumen (Figure 2A). The cells in the narrow cords showed a spindle-shaped morphology with sparse mitotic figures and without nuclear pleomorphism. Red blood cell extravasation with the presence of siderophages, intracytoplasmic incorporation of red blood cells by ECs (autolumination phenomenon), and intra or extracellular hyaline globules, probably corresponding to modified red blood cells, was observed with relative frequency ( Figure 2B,C).  The nodular stage revealed KS spindle cells, which replaced the dermal collagen and presented increased mitotic activity (6-18 mitoses/10 high-power fields) in the anaplastic KS. In the cross-sections, the KS cells appear around small or varying caliber lumens with the intraluminal presence of red blood cells; the lesion adopts a honeycomb-like pattern ( Figure 2D). When longitudinally sectioned, the KS spindle cells showed an elongated nucleus and delimited lumens, virtual or with rows of red blood cells ( Figure 2E). The intracytoplasmic degraded red blood cells and hyaline bodies were present in some KS cells ( Figure 2E).
Nuclear expression for the endothelial marker ERG ( Figure 2F) and for the Herpes virus human 8 (HHV-8) ( Figure 2G) was observed in all cases.
Next, we will pay particular attention to the pre-existing blood vessels and complex structures (associated vessels and skin appendages) in KS lesions and to the newly formed vessels to establish the location, characteristics, relationship and participation of CD34+SCs/TCs in these neovessels.

Pre-Existing (Native) Blood Vessels and Their Relationship with the Inflammatory Infiltrate and Newly Formed Vessels in the Skin Affected by KS in Patch and Plaque Stages
A close relationship between the pre-existing blood vessels, inflammatory infiltrate, and newly formed vessels was observed in KS of the skin. The pre-existing blood vessels, with or without perivascular inflammatory infiltrate, did or did not present perivascular neovessels. Therefore, the pre-existing blood vessels in early cutaneous KS lesions were (a) apparently non-affected (without perivascular inflammatory infiltrate or perivascular neovessels), (b) with perivascular inflammatory infiltrate, and (c) with perivascular neovessels, presenting perivascular inflammatory infiltrate or not. Pre-existing vessels with perivascular inflammatory infiltrate or perivascular neovessels (with or without inflammatory infiltrate) were in similar proportions and predominated over apparently unaffected pre-existing blood vessels.
2.2.1. CD34+SCs/TCs in Pre-Existing Blood Vessels without Perivascular Inflammatory Infiltrate or New Vessels (Apparently Non-Affected Blood Vessels) Double staining for CD34 and αsmooth muscle actin (αSMA) revealed three layers formed by the CD34+ ECs (intima layer), αSMA+ pericytes or vascular smooth muscle cells (media layer), and delimiting CD34+SCs/TCs (external layer) in apparently non-affected pre-existing blood vessels of the two horizontal vascular plexuses and those connecting them in the skin ( Figure 3). Depending on vessel caliber, one or several sheets of delimiting CD34+SCs/TCs were observed ( Figure 3A-D). An exception was the small vessels located in the most superficial areas of the dermal papillae, in which CD34+SCs/TCs were absent. A labyrinthine system of CD34+SCs/TCs was seen between and around the complex structures formed by skin appendages and blood vessels ( Figure 3B,E). In addition to this delimiting arrangement, CD34+SCs/TCs could extend some of their processes into surrounding tissues.

CD34+SCs/TCs in Pre-Existing Blood Vessels with Perivascular Inflammatory Infiltrate
The perivascular inflammatory infiltrate in the pre-existing blood vessels was observed in the space between the CD34+SCs/TCs and pericytes or vascular smooth muscle cells ( Figure 4A). When CD34+SCs/TCs were arranged in several sheets, the inflammatory infiltrate was located between and delimited by them. The inflammatory cells showed expression for CD45 ( Figure 4B), and the inflammatory infiltrate predominated around the vessels in the complex structures ( Figure 4C). ) and complex structures with vessels and glands (bv and gd, respectively, in (B,E)) in areas without perivascular newly formed vessels or inflammatory infiltrate. Three layers in the pre-existing blood vessels are observed in (A,B), with double immunochemistry for CD34 and αSMA. Intima layer, formed by CD34+ ECs (arrowhead) (brown); media layer, made up of αSMA+ pericytes or vascular smooth muscle cells (red); and external layer with CD34+SCs/TCs (arrows) (brown). Similar images in immunofluorescence in (C,E): Intima layer (arrowhead) (green), media layer (red), and external layer with CD34+SCs/TCs (arrows) (green). In (D), CD34 expression is highlighted in both ECs (arrowhead) and CD34+SCs (arrows) by immunofluorescence, with only CD34 (green). Note the formation of a labyrinthine system by  ) and complex structures with vessels and glands (bv and gd, respectively, in (B,E)) in areas without perivascular newly formed vessels or inflammatory infiltrate. Three layers in the pre-existing blood vessels are observed in (A,B), with double immunochemistry for CD34 and αSMA. Intima layer, formed by CD34+ ECs (arrowhead) (brown); media layer, made up of αSMA+ pericytes or vascular smooth muscle cells (red); and external layer with CD34+SCs/TCs (arrows) (brown). Similar images in immunofluorescence in (C,E): Intima layer (arrowhead) (green), media layer (red), and external layer with CD34+SCs/TCs (arrows) (green). In (D), CD34 expression is highlighted in both ECs (arrowhead) and CD34+SCs (arrows) by immunofluorescence, with only CD34 (green). Note the formation of a labyrinthine system by CD34+SCs/TCs around complex structures (blood vessels and glands) in (  The perivascular inflammatory infiltrate in the pre-existing blood vessels was observed in the space between the CD34+SCs/TCs and pericytes or vascular smooth muscle cells ( Figure 4A). When CD34+SCs/TCs were arranged in several sheets, the inflammatory infiltrate was located between and delimited by them. The inflammatory cells showed expression for CD45 ( Figure 4B), and the inflammatory infiltrate predominated around the vessels in the complex structures ( Figure 4C).

CD34+SCs/TCs in Pre-Existing Blood Vessels with Perivascular Neovessels
Neovessels were mainly observed around the pre-existing blood vessels ( Figure 5), predominantly in the dermal, superficial horizontal vascular plexus. A differential fact between the pre-existing blood vessels and neovessels was the presence of αSMA+ pericytes or vascular smooth muscle cells in the former and their absence in the neovessels ( Figure 5).

CD34+SCs/TCs in Pre-Existing Blood Vessels with Perivascular Neovessels
Neovessels were mainly observed around the pre-existing blood vessels ( Figure 5), predominantly in the dermal, superficial horizontal vascular plexus. A differential fact between the pre-existing blood vessels and neovessels was the presence of αSMA+ pericytes or vascular smooth muscle cells in the former and their absence in the neovessels ( Figure 5). . Neovessels (nv, corresponding to type 1) around pre-existing blood vessels (bv). The precise location of the neovessel ECs coincides with that of the CD34+SCs/TCs in the apparently Figure 5. (A-C). Neovessels (nv, corresponding to type 1) around pre-existing blood vessels (bv). The precise location of the neovessel ECs coincides with that of the CD34+SCs/TCs in the apparently non-affected blood vessels or with perivascular inflammatory infiltrate (arrows) (compare with Figures 3 and 4). Note that the pre-existing blood vessels (bv, with αSMA+ pericytes or vascular smooth muscle cells, red) are totally or partially surrounded by the neovessels (without pericytes or vascular smooth muscle cells), giving rise to the image of a vessel within a vessel (promontory sign). Double immunochemistry for CD34 and αSMA. The precise location of the neovessels coincided with that of the previously delimiting CD34+/SCs/TCs around the blood vessels ( Figure 5), suggesting a change from the CD34+SCs/TCs to neovessel ECs. Likewise, intraluminal folds and pillars were observed in the neovessels. Given this, we will first consider the main types and characteristics of the initial neovessels, and of the intraluminal folds and pillars, paying particular attention to the findings supporting their formation from delimiting CD34+SCs/TCs. Next, we will briefly consider the evolution of the neovessels in the advanced stages of KS.

Initial Neovessels in Early KS Lesions
Although several patterns of neovessels were present in the patch and plaque stages of KS, two main types were seen (types 1 and 2) ( Table 1). Both types of neovessels were predominantly arranged around pre-existing blood vessels, coinciding in a location with that previously held by CD34+SCs/TCs (Figures 5 and 6A-C), as mentioned above. Next, we consider the characteristics of type 1 and 2 neovessels. Type 1 neovessels showed lumens of differing sizes and configurations, ranging from slit-like vascular spaces to wide sacs with a lymphangiomatous appearance ( Figure 5). Generally, the different neovessel lumens converged, giving rise to paths that varied in aspect. The lumens were lined by a monolayer of flattened ECs, which showed expression for markers of both blood and lymphatic vessels ( Figures 5 and 6). Thus, in addition to positivity for CD34 (a marker for blood vessel ECs) ( Figures 5 and 6A), the ECs of the neovessels also expressed D2-40 (a marker for lymphatic vessel ECs) ( Figure 6B,C), while the ECs of the pre-existing vessels were positive for CD34 ( Figures 5 and 6A) and negative for D2-40 ( Figure 6B,C). The difference in marker expression in the ECs, according to blood or lymphatic vessels, was evident when each marker was combined with αSMA expression, as indicated above. Likewise, when present, the CD45+ perivascular inflammatory cells were distinguished by double immunostaining (for D2-40 and CD45) ( Figure 6C). Ultrastructurally, neovessel ECs showed alternating thick and thin regions, resembling the podomeres and podoms of telocytes, respectively ( Figure 6D and compare this with the insert). Discontinuity zones, small intercellular junctions and cell processes extending to the interstitium were also observed in neovessel ECs ( Figure 6D). Pillars and folds, with a cover and a core, were present in the lumen of neovessels. The cover of pillars and folds was formed by ECs with the same characteristics as those lining the neovessels, and the core contained packed collagen fibers, which were positive for collagen I ( Figure 7A). Nascent pillars formed by ECs were also seen ( Figure 7A). The co-expression of D2-40 y Lyve-1 was observed in some neovessels ( Figure 7B), and CD31 expression was seen in the ECs of both pre-existing blood vessels and neovessels. The core of larger intraluminal folds could present blood vessels with or without perivascular inflammatory infiltrate (blood vessels within neovessels, the promontory sign) ( Figure 7C). Type 1 neovessels showed lumens of differing sizes and configurations, ranging from slit-like vascular spaces to wide sacs with a lymphangiomatous appearance ( Figure 5) Generally, the different neovessel lumens converged, giving rise to paths that varied in aspect. The lumens were lined by a monolayer of flattened ECs, which showed expression and folds was formed by ECs with the same characteristics as those lining the neovessels, and the core contained packed collagen fibers, which were positive for collagen I ( Figure  7A). Nascent pillars formed by ECs were also seen ( Figure 7A). The co-expression of D2-40 y Lyve-1 was observed in some neovessels ( Figure 7B), and CD31 expression was seen in the ECs of both pre-existing blood vessels and neovessels. The core of larger intraluminal folds could present blood vessels with or without perivascular inflammatory infiltrate (blood vessels within neovessels, the promontory sign) ( Figure 7C).

Characteristics of Type 2 Neovessels
Type 2 neovessels were more numerous in the plaque stage and appeared located around blood vessels in the previous location of the CD34+SCs/TCs, as occurred in type 1 vessels ( Figure 8A-D). Most type 2 neovessels presented virtual or small lumens and a fascicular appearance when sectioned longitudinally ( Figure 8B-D). Depending on the orientation and plane of sectioning, their ECs had a spindle or oval morphology and were generally more voluminous than those of type 1 vessels. Type 2 neovessel ECs also expressed markers for ECs of both blood and lymphatic vessels. With varying frequencies, one or several red blood cells were observed in the small lumen of these neovessels ( Figure 8E,F). The red blood cells appeared, forming rows when the vessels were sectioned longitudinally ( Figure 8E). Neovessels with more dilated lumens and presenting with numerous intra-and extraluminal red blood cells and the occasional pillars were intermingled with those with smaller or virtual lumens and were occasionally numerous. Ultrastructurally, some of these neovessels showed virtual lumens, which were not demonstrated by other procedures ( Figure 9A). The ECs of type 2 neovessels with discontinuities, different electron densities and small interendothelial adherent and peg-and-socket junctions were also frequently observed under the electron microscope ( Figure 9A-C).   In addition to the coincidence of the neovessel EC location and that of delimiting CD34+SCs/TCs, the following findings were observed during neovessel formation. The somatic region and processes of one or more delimiting CD34+SCs/TCs folded or converged, forming occasional, isolated small vessel lumens in the early stages of KS ( Figure  10A-C). These small neovessel lumens alternated with other perivascular CD34+SCs/TCs,

Findings Supporting CD34+SC/TC Participation in the Origin of Neovessel ECs
In addition to the coincidence of the neovessel EC location and that of delimiting CD34+SCs/TCs, the following findings were observed during neovessel formation. The somatic region and processes of one or more delimiting CD34+SCs/TCs folded or converged, forming occasional, isolated small vessel lumens in the early stages of KS ( Figure 10A-C). These small neovessel lumens alternated with other perivascular CD34+SCs/TCs, without forming lumens. The number of neoformed vessel lumens from the CD34+SCs/TCs appeared to increase around other pre-existing blood vessels ( Figure 10D). These newly formed vessel lumens could be subdivided by intraluminal CD34+SCs/TCs endowed with two or several cytoplasmic processes, which extended to different points of the neovessel wall ( Figure 10E). The spaces formed in the lumen of the neovessels could also be separated by voluminous CD34+ ECs or by their processes (Figure 10E and insert).
The ECs of the initially evolved neovessels gave rise to enlarged and confluent lumens (neovessel type 1) or formed narrow cords (neovessel type 2) and showed expression for markers of both blood and lymphatic ECs. A similar expression for both types of markers was observed in the CD34+SCs/TCs interposed between the neovessels and without forming a lumen ( Figure 11A-C). Although these neovessels could appear separated from the pre-existing blood vessels, well-oriented histological sections showed their relationship with the latter ( Figure 11D). Likewise, the ECs of neovessels were observed in continuity with delimiting CD34+SCs/TCs arranged between pre-existing blood vessels ( Figure 11E). As mentioned, the ECs of the pre-existing blood vessels did not show expression for lymphatic vessel markers, unlike the neovessels ( Figure 11A,C).

CD34+SCs/TCs during the Formation of Pillars in the Lumen of Neovessels
Some of the transformed CD34+SCs/TCs lining the neovessels were observed as preserving one or two of their processes that extended into the surrounding tissues ( Figure 12A1-3). These processes were seen between and around collagen fibers, which they partially or totally surrounded. The surrounding processes, together with the enveloping collagen, could also appear contacting two points of the neovessel wall ( Figure 12A4) or being incorporated into the neovessel lumen, acquiring a pillar aspect ( Figure 12A5). These pillars were numerous in some neovessels ( Figure 12B,C), and their covering cells could present Ki-67 nuclear expression (insert Figure 12B).

Evolution of the Neovessels in KS
Dilated type 1 neovessels, generally with numerous intraluminal folds and pillars (papillae), were observed in the lymphangiectatic and lymphangiomatous variants of KS (an increase of the already described findings for type 1 vessels). Frequently, the folds in the neovessels contained pre-existing blood vessels (a vessel inside a vessel or a promontory sign). The lumen of these type 1 neovessels appeared empty or with some inflammatory cells and/or occasional red blood cells.
Numerous neovessels, predominantly type 2, interposed the pre-existing blood vessels, and intra-and extraluminal red blood cells were usual components in the nodular stage of KS ( Figure 13A-C). Intracytoplasmic incorporation of degraded blood cells by the ECs was frequent ( Figure 13D). Some mononuclear inflammatory cells and scarce collagen material were also seen.

Observations through Serial Histologic Sections
In the serial histologic sections with different immunomarkers in each, the following findings were specified in type 1 neovessels (Figure 14, compare A and B, Table 1), including their location around blood vessels, similar to that of the delimiting CD34+SCs/TCs; their constant expression of markers for both blood and lymphatic vessels, different from that of pre-existing blood vessels; the presence of evident vascular spaces alternating with slit-like vascular lumens, as well as with CD34+SCs/TCs, forming small lumens or not; the existence of discontinuities in the neovessel lining; the formation of intravascular folds centered by collagen; and the convergence of neovessels that appear isolated in other histologic sections. Vessels within vessels were observed when wide neovessels converged around pre-existing blood vessels (a promontory sign) ( Figure 15A,B). Likewise, in adjacent sections, the appearance and disappearance of pillars (hallmarks of intussusceptive angiogenesis) were demonstrated ( Figure 15C,D). Serial sections, more easily obtainable using conventional techniques with hematoxylin-eosin staining, showed longer neovessel pathways arranged around the pre-existing vessels, nerves and glands, presenting regions that appeared isolated and/or modified in other sections ( Figure 15E-G). The predominant presence of small or virtual lumens in the elongated type 2 neovessels around pre-existing blood vessels, expressing markers for ECs of both blood and lymphatic vessels, was also demonstrated in the serial sections ( Figure 15H,I, Table 1). without forming lumens. The number of neoformed vessel lumens from the CD34+SCs/TCs appeared to increase around other pre-existing blood vessels ( Figure 10D) These newly formed vessel lumens could be subdivided by intraluminal CD34+SCs/TCs endowed with two or several cytoplasmic processes, which extended to different points of the neovessel wall ( Figure 10E). The spaces formed in the lumen of the neovessels could also be separated by voluminous CD34+ ECs or by their processes (Figure 10E and insert).  out forming a lumen ( Figure 11A-C). Although these neovessels could appear se from the pre-existing blood vessels, well-oriented histological sections showed t lationship with the latter ( Figure 11D). Likewise, the ECs of neovessels were obse continuity with delimiting CD34+SCs/TCs arranged between pre-existing blood ( Figure 11E). As mentioned, the ECs of the pre-existing blood vessels did not s pression for lymphatic vessel markers, unlike the neovessels ( Figure 11A,C). tially or totally surrounded. The surrounding processes, together with lagen, could also appear contacting two points of the neovessel wa being incorporated into the neovessel lumen, acquiring a pillar asp These pillars were numerous in some neovessels ( Figures 12B,C), and could present Ki-67 nuclear expression (insert Figure 12B). tory cells and/or occasional red blood cells. Numerous neovessels, predominantly type 2, interposed the pre-existing blood vessels, and intra-and extraluminal red blood cells were usual components in the nodular stage of KS ( Figure 13A-C). Intracytoplasmic incorporation of degraded blood cells by the ECs was frequent ( Figure 13D). Some mononuclear inflammatory cells and scarce collagen material were also seen.

Discussion
In KS of the skin, we contribute (a) the participation of the delimiting CD34+SCs/TCs in the external layer of pre-existing blood vessels and around skin appendages in the formation of neovessels, behaving as resident mesenchymal/stromal cells capable of transdifferentiation into neovessel ECs and (b) the formation of two main types of initial neovessels in the early stages of KS, leading to progressive sarcomatous lesions.
The distinction between pre-existing blood vessels and initial neovessels was important to our observations on the evolution of delimiting CD34+SCs/TCs during their transformation into neovessel ECs in the early KS lesions. This distinction was based on the expression in their ECs of only markers for blood vessels and the presence of prominent αSMA+ mural cells (pericytes or vascular smooth muscle cells) in pre-existing blood vessels versus the expression of markers for ECs of both blood and lymphatic vessels and the absence of mural cells in the initial neovessels.
The main events of CD34+SC/TC participation in the formation of neovessels and the evolution of the latter in the KS stages can be summarized as follows ( Figure 16): (1) In the very early stages of KS, CD34+SCs/TCs retain their characteristics in the external layer of pre-existing blood vessels and around skin appendages, with and without perivascular inflammatory infiltrates, which, when present, they encompass. (2) Small lumens are formed between semi-detached and folded CD34+SCs/TCs. (3) Predominantly around pre-existing blood vessels, the lumens increase in number, converge, and form evident neovessels, whose lining ECs express markers for both blood and lymphatic vessels (the formation of neovessels around pre-existing blood vessels). In this phase, transitional findings between some neovessel neighboring CD34+SCs/TCs and neovessel ECs are seen, including (a) a presence in the lumen of neovessels of stellate CD34+SCs/TCs contacting the neovessel wall, (b) neighboring CD34+SCs/TCs to neovessels showing markers for neovessel ECs, (c) a sharing of some ultrastructural characteristics of CD34+SCs/TCs by neovessel ECs (e.g., processes toward the interstitium, alternating thick and thin cytoplasmic regions and discontinuities between some of the cells lining the neovessels), and (d) the replacement of CD34+SCs/TCs around pre-existing blood vessels by lining neovessel ECs. (4) Two main types of initial neovessels are formed: types 1 and 2, respectively, showing (a) ECs with flattened or spindle-shaped morphology, (b) anastomosing irregular pathways, or an elongated and fascicular appearance, (c) lumens of varying size, generally empty or very small, with some red blood cells, and (d) intraluminal folds and mature pillars (papillae), or only intraluminal endothelial bridges (nascent pillars). (5) Type 1 neovessels reach their greatest expression in the lymphangiectatic and lymphangiomatous variants of KS, while type 2 does so in KS lesions with a sarcomatous appearance. Next, we consider these events.
For the transitional events between the CD34+SCs/TCs and ECs, the following issues should be considered: (a) The intraluminal persistence in the neovessels of stellate CD34+SCs/TCs keeping attachment points of their processes with the lining neovessel cells is compatible with a mechanism of vessel lumen formation by separation of adjacent CD34+SCs/TCs. In addition, the intraluminal CD34+SCs/TCs and their multiple or bipolar processes can also participate in the creation of new intraluminal spaces in the neovessels, increasing neovessel numbers by vessel splitting (intussusceptive angiogenesis). Therefore, the partial separation of adjacent and contacting CD34+SCs/TCs is an additional mechanism to those previously proposed in intussusceptive angiogenesis [58][59][60][61][62][63][64][65]. (b) The coincident expression of markers for the blood and lymphatic vessels in neovessel ECs, and in some neighboring and interspersed CD34+SCs/TCs during neovessel formation, coincides with the ability of the latter to gain new markers [22,[34][35][36][37] and in this case for ECs, of blood and lymphatic vessels (c). While the existence of some neovessel ECs retaining the CD34+SC/TC characteristics supports the transition from one cell type to another, it is also an exponent of the defining ultrastructural signs of the telocytes [20,21]. (d) The total replacement of CD34+SCs/TCs around pre-existing blood vessels with neovessel ECs could be due to CD34+SC/TCs' participation as progenitor cells or simply to their loss [31]. The absence of the degenerative signs in CD34+SCs/TCs during neovessel formation is incompatible with the latter possibility. This replacement is, therefore, another indication for their participation in neovessel formation.
The set of events outlined above supports previous studies by other authors on the participation of mesenchymal/stromal cells in the origin of KS cells [14][15][16][17] and establishes the precise location and characteristics of the resident cells (delimiting CD34+SCs/TCs) with this mesenchymal capacity. However, the fact that CD34+SCs/TCs are a source of neovessel ECs in KS does not exclude the participation of ECs in this origin, mainly of lymphatic vessels, which is also supported by the immunoprofile of neovessel ECs [6,7]. The origin of different progenitor cells concurs with the evidence for multiclonality in KS [3,4] and would lead to the connection of and interrelationship between the CD34+SCs/TCs and blood and lymphatic ECs during neovessel development. The CD34+SC/TC and EC connections with pre-existing blood vessels could facilitate the extravasation of red blood cells with hemosiderosis (abnormal neovessels connecting to blood vessels). However, one or several extravasated red blood cells could be surrounded by the CD34+SCs/TCs as described for the neoplastic cells of lobular carcinoma of the breast, in which the surrounding CD34+SCs/TCs originate cancer-associated fibroblasts [66]. Likewise, the edema retained by the CD34+SCs/TCs around pre-existing blood vessels could be partially removed by connecting lymphatic vessels (KS-associated edema). All these issues require further studies.
The formation of two main types of initial neovessels and the predominance of one in lesions with lymphangiomatous characteristics and of the other with a sarcomatous aspect during KS evolution suggests it is related to lesion variants.
Papillae in vessel lumens have been described in several tumors and pseudotumors of blood and lymphatic vessels [67][68][69][70][71][72], including KS lesions [18,19]. In previous works, we have demonstrated the similarity between these papillae and the folds and pillars that are the hallmarks of intussusceptive angiogenesis and lymphangiogenesis [63,64,66,73,74], highlighting their role in the growth of vascular networks and the morphogenesis of vessel tumors/pseudotumors [66,70,74]. In this case, CD34+SCs/TCs and their processes surrounding collagen fibers separate from the interstitium, travel into the vessel lumen and form the cover and core of folds and pillars. This mechanism resembles that described for ECs and collagen as inverse sprouting [65]. When the intraluminal fold cores contain blood vessels, with or without perivascular inflammatory infiltrates, they appear as vessels within vessels, which can explain the promontory sign described in some types of KS lesions [18,19,75,76].
This work has a histogenic and potential clinical and therapeutic interest. Indeed, it contributes to a better knowledge of the type and location of resident stromal precursor cells with a vasoformative capacity in KS, which could make it easier to obtain precursor cells for in vitro studies and assess their modulation during tumor development. It could also be the basis for future studies that confirm the possible evolution of the early lesions toward the different variants of KS and, therefore, their prognostic projection. These studies require larger series of cutaneous KS cases to monitor histological lesions over time, patient evolution and the application of new techniques, such as histomorphometric analysis, which other authors have used on telocytes [77,78].

Human Tissue Samples
The archives of Histology and Anatomical Pathology of the Departments of Basic Medical Sciences of La Laguna University, University Hospital, and Eurofins ® Megalab-Hospiten Hospitals of the Canary Islands were searched for cases of KS. Specimens (paraffin blocks) were obtained from 49 cases. The patients were Caucasian: 28 males and 21 females, aged 17-88 years. All specimens were studied by conventional histologic techniques using hematoxylin-eosin staining to select blocks and more demonstrative areas in the histological sections. Of them, 30 cases were used for the immunochemistry procedures and immunofluorescence in confocal microscopy, and four cases were used for electron microscopy. A combination of different immunomarkers in the immunohistochemistry and immunofluorescence procedures and ultrastructural observations were used to establish the general characteristics of the CD34+SCs/TCs and to assess their participation in neovessel formation. Serial histological sections with different immunomarkers in each, including the observations in confocal microscopy, were undertaken to confirm the previously suggested findings. Ethical approval for this study was obtained from the Ethics Committee of La Laguna University (Comité de Ética de la Investigación y de Bienestar Animal, CEIBA 2022-3192), including the dissociation of the samples from any information that could identify the patient. The authors, therefore, had no access to identifiable patient information.

Light Microscopy
The specimens were fixed in a buffered neutral 4% formaldehyde solution, embedded in paraffin, and cut into 3 µm-thick sections. The sections were deparaffinized, hydrated and stained with haematoxylin and eosin (H&E).

Electron Microscopy
Electron microscopy for the ultrastructural study was carried out, as described previously [37]. The specimens were initially fixed in 2% glutaraldehyde in 100 mM sodium cacodylate buffer, pH 7.4, for 6 h at 4 • C, and subsequently, washed in the same buffer, post-fixed for 2 h in 1% osmium tetroxide, dehydrated through increasing concentrations of ethanol (40-100%) and embedded in Spurr's resin. Ultrathin sections were collected on nickel grids, double stained with uranyl acetate and lead citrate and examined with the JEOL ® 100B and JEM 1011 Akishima, Tokyo, Japan, electron microscopes.

Quantitative Analysis
Quantitative analysis of the neovessels was performed in cases of KS in which immunochemistry (D2-40) was used (n: 30). The percentages in neovessels type 1 and type 2 are reported as a mean of each group and +/− standard deviation, and the early and advanced stages were analyzed by the Student's t-test, considering a statistically significant p-value of less than 0.05.

Conclusions
In conclusion, in KS of the skin, we demonstrate that CD34+SCs/TCs, mainly in the external layer of pre-existing blood vessels, are resident mesenchymal cells capable of transdifferentiation into ECs. We establish: (a) the transitional findings between both types of cells, including the presence of CD34+SCs/TCs expressing markers for the ECs of blood and lymphatic vessels and the replacement of CD34+SCs/TCs by the EC of neovessels; (b) the possible interrelationships of the CD34+SC/TC descendants with ECs from other sources; (c) the formation of two types of neovessels and their evolutionary phenomena, suggesting their role in the origin of the different variants of KS with lymphangiomatous or spindle-cell patterns; and (d) the subsequent neovessel growth by mechanisms of intussusception (intussusceptive angiogenesis and lymphangiogenesis), all of which are of histogenic, clinical, and therapeutic interest.